Novel glycan conjugates and methods of use thereof

ABSTRACT

The present disclosure is directed to vaccines, antibodies, and/or immunogenic conjugate compositions targeting the SSEA3/SSEA4/GloboH associated epitopes (natural and modified) which elicit antibodies and/or binding fragment production useful for modulating the globo-series glycosphingolipid synthesis. The present disclosure relates to methods and compositions which can modulate the globo-series glycosphingolipid synthesis. Particularly, the present disclosure is directed to glycoenzyme inhibitor compound and compositions and methods of use thereof that can modulate the synthesis of globo-series glycosphingolipid SSEA3/SSEA4/GloboH in the biosynthetic pathway; particularly, the glycoenzyme inhibitors target the alpha-4GalT; beta-4GalNAcT-I; or beta-3GalT-V enzymes in the globo-series synthetic pathway. Moreover, the present disclosure is also directed to the method of using the compositions described herein for the treatment or detection of hyperproliferative diseases and/or conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/832,993, filed Aug. 21, 2015, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application No. 62/107,378,filed on Jan. 24, 2015, entitled NOVEL GLYCAN CONJUGATES AND METHODS OFUSE THEREOF, the contents of which is hereby incorporated by referenceas if set forth in its entirety.

FIELD

The present disclosure relates to methods and compositions which canmodulate the globo-series glycosphingolipid synthesis. Particularly, thepresent disclosure is directed to glycoenzyme inhibitor compound andcompositions and methods of use thereof that can modulate the synthesisof globo-series glycosphingolipid SSEA3/SSEA4/GloboH in the biosyntheticpathway; particularly, the glycoenzyme inhibitors target thealpha-4GalT; beta-4GalNAcT-I; or beta-3GalT-V enzymes in theglobo-series synthetic pathway. Additionally, the present disclosure isalso directed to vaccines, antibodies, and/or immunogenic conjugatecompositions targeting the SSEA3/SSEA4/GloboH associated epitopes(natural and modified) which can elicit antibodies and/or bindingfragment production useful for modulating the globo-seriesglycosphingolipid synthesis. Moreover, the present disclosure is alsodirected to the method of using the compositions described herein forthe treatment or detection of hyperproliferative diseases and/orconditions.

BACKGROUND OF THE INVENTION

The carbohydrate antigens GloboH, stage-specific embryonic antigen-3(SSEA3), and stage-specific embryonic antigen-4 (SSEA4) are closelyrelated to one another in either structure or in function. GloboH, SSEA3and SSEA4 are globo-series glycosphingolipids, with SSEA3 being thenon-fucosylated pentasaccharide precursor structure of GloboH, SSEA4 issialylated SSEA3 with sialic acid a2-3 links to the non-reducing end ofgalactose of SSEA3.

Stage-specific embryonic antigen-3 (SSEA3) was first identified anddefined by the reactivity of an IgM monoclonal antibody generated in arat immunized with 4- to 8-cell stage mouse embryos. This monoclonalantibody reacted with all mouse preimplantation embryos from oocytes upto the early blastocyst stage where its expression became morerestricted, in the primitive endoderm after implantation. The SSEA3antigenic determinant was determined to be a carbohydrate present onglycolipids and glycoproteins; it was also found on humanteratocarcinoma cells and human erythrocytes. In a panel of structuresisolated from the 2102Ep human teratocarcinoma cell line, the SSEA3antibody had the highest affinity for Galβ(1-3)GalNAcβ(1-3)Galα(1-4)Galβ(1-4)Glcβ(1)Cer. This structure is also known as Gb5,galactosyl-globoside, or globopentaosylceramide.

Synthesis of SSEA3 occurs when 31,3-_(g)alactosyltransferase V(β3GalT-V) transfers galactose to the GalNAc of globoside to form Gb5 orgalactosyl-globoside. It was determined that SSEA3 was not expressed inhematopoietic or mesenchymal stem cells. Based on immortalized lymphnode lymphocytes from primary lung cancer patients, generatedhybridomas, and selected for antibody secreting clones; monoclonalantibodies were then generated from two of these clones—J309 and D579,which recognized the SSEA3 antigenic determinant. The antibodiesrecognized SSEA3 on several tumor cell lines including lung and breastcancer cell lines, and a teratocarcinoma cell line; in an immuneadherence assay, rodent monoclonal SSEA3 antibody, also referred to asMC631, reacted against the same cell lines as the J309 and D579antibodies. SSEA3 has also been found on testicular germ cell tumors, aswell as in breast cancer and in BCSCs (breast cancer stem cells).

Chang et al. looked at SSEA3 expression on normal tissues using a tissuemicroarray because its location outside of cancer and development waslargely unknown. The group found SSEA3 to be expressed on normalepithelium of colon, esophagus, small intestine, kidney, prostate,rectum, skin, testis, thymus, and uterine cervix. Expression was locatedonly on the apical surfaces of epithelial cells or in the cytoplasm,which are considered immune system restricted or inaccessible sites. Inan experiment using a KLH conjugated GloboH monovalent vaccine in mice,an antibody response was made to only the GloboH antigen. When α-GalCerwas added as an adjuvant, the amount of overall antibody productionincreased and the mice made polyclonal antibodies to both the GloboH,the SSEA3 and the SSEA4 antigen structures, which vaccination was unableto generate in the absence of the adjuvant. This result showed thatSSEA3, GloboH and SSEA4 could make promising targets for cancer vaccinesand could be targeted simultaneously.

However, most tumor associated carbohydrate antigens have poorimmunogenicity and many approaches have been developed to increase theimmune response of carbohydrate-based vaccines, including conjugationwith a carrier protein,administration with an immunologic adjuvant usingunnatural glycosidic linkage, clustered antigens, unimolecularpolyvalent vaccine or hetero-glycan multivalent vaccine. Using thesestrategies, a few carbohydrate-based vaccines that could elicitsignificant immune responses to target glycan structures were designedfor cancer therapy and entered clinical trials. Among them, the clinicaltrials of Theratope and GMK with adjuvant QS-21 failed to producestatistically significant difference between time-to-disease and overallsurvival rate. Mot likely these two vaccines could not elicit robust Tcell-dependent immune response in patients. Specifically, Theratope andGMK induced a higher level of IgM in patients but could not induce astrong immune IgG response, which is a major problem incarbohydrate-based vaccine development.

Previous studies showed that modification of carbohydrate antigenstructures (MCAS) could effectively elicit a higher level of immuneresponse. For example, in the modification study of the capsularpolysaccharide of group B meningococci, the N-acetyl groups ofα-(2,8)-linked polysialic acid (PSA) was replaced with the N-propinoylgroup and such a modification elicited a high antibody response torecognize not only the N-propinoyl PSA, but also the nature N-acetylPSA. Similar approaches were applied to STn and GM3 antigens to producehigh antibody titers against modified and nature forms. The resultsindicated that N-phenylacetyl, N-fluoroacetyl or N-difluoroacetylmodifications on glycan antigens could improve the immunogenicity.Moreover, the Schultz group reported that incorporation of ap-nitrophenylalanine into the tumor necrosis factor-α (TNF-α) couldbreak immune tolerance and induce more antibody response to TNF-α. Usingglycans as antigens, although some progress has been achieved, mostcases are the N-modification of disaccharide (STn), trisaccharide (GM3)and polysialic acid (PSA) and some are based on fluorinated MUC1glycopeptide antigens.

SUMMARY OF THE INVENTION

The present disclosure is based on the discovery that the modificationof the stage-specific embryonic antigens (SSEA3 and SSEA4) with certaingroups disclosed herein elicited robust IgG antibody response tospecifically recognize SSEA3 and SSEA4, respectively. The antibodiesinduced by an immunogenic composition comprising such unnatural glycanmoiety are able to mediate the complement-dependent cell cytotoxicityagainst tumor cells.

Accordingly, the present invention features the design of antibodiesagainst SSEA3 and/or SSEA4 for treating cancers. The present inventionalso features novel compounds consisting of the modified carbohydrateantigens (SSEA3 and SSEA4), glycan conjugates comprising such, andimmunogenic compositions and vaccines thereof.

The present disclosure also provides methods of using synthetic glycanconjugates described herein to treat or reduce hyperproliferativedisease such as cancer.

Additionally, the present disclosure is also directed to vaccines and/orimmunogenic conjugate compositions targeting the SSEA3/SSEA4/GloboHassociated epitopes (natural and modified) which can elicit antibodiesand/or binding fragment production useful for modulating theglobo-series glycosphingolipid synthesis. Moreover, the presentdisclosure is also directed to the method of using the compositionsdescribed herein for the treatment or detection of hyperproliferativediseases and/or conditions.

Accordingly, the present invention features the design of antibodiesagainst SSEA3 for treating cancers. The present invention also featuresnovel compounds consisting of the modified carbohydrate antigens (SSEA3,SSEA4), glycan conjugates comprising such, and immunogenic compositionsand vaccines thereof

In one aspect, the present invention provides a compound of formula (I):

or a salt thereof, wherein X₁, R¹, R², R³, R⁴, R⁵, R⁶ and L are asdescribed herein. In certain embodiments, a compound of Formula (I) isuseful for making an immunogenic composition for treating cancers.

In another aspect, the present invention provides a compound of Formula(II):

or a salt thereof, wherein X₁, R¹, R², R³, R⁸, R⁹, R¹⁰, R¹¹, and R_(N)are as described herein. In certain embodiments, a compound of Formula(II) is useful for making an immunogenic composition for treatingcancers.

In another aspect, the present invention provides an immunogeniccomposition, comprising (a) a glycan conjugate including a carrier andone or more glycans, and optionally (b) an adjuvant,

wherein: each of the one or more glycans is conjugated with the carrierthrough a linker, having the formula (III) or (IV):

wherein X₁, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, and R_(N) are asdescribed herein.

In certain aspects, it is contemplated that any construct of vaccinecontaining a combination of any one or more of the three glycans (SSEA3,SSEA4 and GloboH) and analogs thereof in any ratio can be linked to acarrier.

wherein n can be an integer from 1 to 10;

wherein Glycan can be selected from the group consisting of Formulas I,II, III, and IV;

wherein if n is 2 or more, each Glycan can be the same as another Glycanon the aspartyl peptide or a difference Glycan on the aspartyl peptide.

In some embodiments, Glycan can be selected from the group consisting ofSSEA3, SSEA4, and GloboH.

In some embodiments, the exemplary multivalent construct can be:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and L on each glycan moiety can be thesame or different.

In certain embodiments, the immunogenic composition of the inventioncomprises an adjuvant. Exemplary adjuvants suitable for the inventionare as described herein.

In certain embodiments, the immunogenic composition is capable ofeliciting an immune response against a cancer cell in a subject. Incertain embodiments, the cancer cell is selected from the groupconsisting of a brain cancer cell, a lung cancer cell, a breast cancercell, an oral cancer cell, an esophageal cancer cell, a stomach cancercell, a liver cancer cell, a bile duct cancer cell, a pancreatic cancercell, a colon cancer cell, a kidney cancer cell, a bone cancer cell, askin cancer cell, a cervical cancer cell, an ovarian cancer cell, and aprostate cancer cell.

In certain embodiments, the immune response includes generation ofantibodies that specifically bind to one or more of the antigensselected from the group consisting of GloboH, SSEA3 and SSEA4. Incertain embodiments, the antibodies are developed to target one or moreof GloboH, SSEA3 and SSEA4 expressed on the surface of cancer cells orcancer stem cells , and trigger CDC and/or ADCC to kill these cells. Incertain embodiments, the antibodies predominantly include IgGantibodies. In certain embodiments, the immunogenic compositionsprovided herein mainly induce IgG1, IgG2b, IgG2c and IgG3.

Further, the present disclosure features monoclonal antibodies andbinding fragments raised against the immunogenic composition describedherein.

In one embodiment, the antibody is a human antibody.

In one embodiment, the antibody is a humanized antibody.

In one embodiment, the antibody is specifically targeted against one ormore of SSEA4, SSEA3, or GloboH.

In one embodiment, the antibody is specifically targeted against SSEA3.

In one embodiment, the antibody is specifically targeted against SSEA4.

In one embodiment, the antibody is a homogeneous antibody having thebiantennary glycan terminated by two sialic acid in alpha-2,6-linkage.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising an effective amount of the antibody orantigen-binding fragment specifically targeted against one or more ofSSEA4, SSEA3, or GloboH and a pharmaceutically acceptable carrier

In one embodiment, the pharmaceutical composition comprises acombination of antibodies and/or binding fragment thereof eachindependently targeting one or more of the SSEA4, SSEA3, and/or GloboHglycans.

In one embodiment, the pharmaceutical composition is useful for thetreatment of cancer, infectious diseases, and/or anti-inflammatorydiseases,

In one embodiment, the pharmaceutical composition comprises antibodiesor binding fragments thereof having universal biantennary N-glycanterminated with sialic acid in alpha-2,6-linkage.

In another aspect, the present invention provides a cancer vaccinecomprising an immunogenic composition described herein and apharmaceutically acceptable excipient.

In another aspect, the present invention provides methods for treatingand/or reducing the risk for cancer in a subject comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an immunogenic composition or a cancer vaccine as describedherein.

The treatment results in reduction of tumor size, elimination ofmalignant cells, prevention of metastasis, prevention of relapse,reduction or killing of disseminated cancer, prolongation of survivaland/or prolongation of time to tumor cancer progression.

In some embodiments, the treatment further comprises administering anadditional therapy to the subject prior to, during or subsequent to theadministering of the immunogenic composition or the cancer vaccinedescribed herein. In some embodiments, the additional therapy istreatment with a chemotherapeutic agent. In some embodiments, theadditional therapy is radiation therapy.

Another aspect of the present disclosure features a method ofvaccinating a mammal against cancers, comprising administering to themammal a pharmacologically effective amount of an immunogeniccomposition or a cancer vaccine as described herein.

In some embodiments, the mammal is a human. In some embodiments, theimmunogenic composition or the cancer vaccine described herein isadministered subcutaneously.

Examples of the cancer include, but are not limited to, brain cancer,lung cancer, breast cancer, oral cancer, esophagus cancer, stomachcancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer,kidney cancer, cervix cancer, ovary cancer and prostate cancer. In someembodiments, the cancer is brain cancer, lung cancer, breast cancer,ovarian cancer, prostate cancer, colon cancer, or pancreas cancer.

In another aspect, the present invention provides methods ofsynthesizing the compounds of the invention as described herein.

In yet another aspect, the present disclosure features the process formaking an immunogenic composition or a cancer vaccine as describedherein.

The details of certain embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the Detailed Description, the Figures, the Examples, andthe Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Biosynthetic Pathway of Globo Series of Glycosphingolipids.

FIG. 2: The induced GloboH-IgG collected from different epitope ratiosof SSE4-CRM197 or SSEA4-Gc-CRM197 immunization.

FIG. 3A: Native SSEA4, as well as all eight SSEA4 analogs, could elicitIgG antibodies against SSEA4 when combining the use of Gal-C34.

FIG. 3B: Native SSEA4, as well as all eight SSEA4 analogs, could elicitIgM antibodies against SSEA4 when combining the use of Gal-C34.

FIG. 4A: Native SSEA4, as well as all eight SSEA4 analogs, could elicitIgG antibodies against SSEA4 when combining the use of Glc-C34.

FIG. 4B: Native SSEA4, as well as all eight SSEA4 analogs, could elicitIgM antibodies against SSEA4 when combining the use of Glc-C34.

FIG. 5: The glycan-protein conjugation method affects the immuneresponse.

DETAILED DESCRIPTIONS

The present disclosure is based on the surprising discovery that themodification of the stage-specific embryonic antigens (SSEA3 and SSEA4)with certain groups elicited robust IgG antibody response tospecifically recognize SSEA3 and SSEA4, respectively.

In some examples, the modification of SSEA3 comprises a fluoro, an azidoor an O-phenyl group at the one or more positions of the glucose ofSSEA3. In some examples, the modification of SSEA3 comprises a fluoro,an azido or an O-phenyl group at the one or more positions of thenon-reducing end galactose. In some examples, the modification of SSEA4comprises a fluoro, an azido or an O-phenyl group at one or morepositions of the glucose of SSEA4. In some examples, the modification ofSSEA4 comprises a fluoro, an azido or an O-phenyl group at one or morepositions of the sialic acid residue.

Described herein are SSEA3 and SSEA4 analogs having the modification atthe reducing and/or non-reducing end. Such SSEA3 and SSEA4 analogs canelicit a stronger immune response (e.g., induction of IgG antibodiesagainst SSEA3 and/or SSEA4) as compared to the native SSEA3 and SSEA4.The antibodies induced by an immunogenic composition comprising suchunnatural glycan moiety are able to mediate the complement-dependentcell cytotoxicity against tumor cells.

Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5th Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3rd Edition, CambridgeUniversity Press, Cambridge, 1987. Moreover, exemplary glycan andantibody methodologies are described in Wong et al, US20100136042,US20090317837, and US20140051127, the disclosures of each of which arehereby incorporated by reference.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C1-6” is intended toencompass C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6,C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C1-20 alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”).Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n-propyl(C3), iso-propyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4),iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl(C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6).Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8)and the like.

Unless otherwise specified, each instance of an alkyl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C1-10 alkyl (e.g., —CH3). In certain embodiments, thealkyl group is substituted C1-10 alkyl.

“Alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C2-20 alkenyl”). Insome embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-10alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl(C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like.Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenylgroups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and thelike. Additional examples of alkenyl include heptenyl (C7), octenyl(C8), octatrienyl (C8), and the like. Unless otherwise specified, eachinstance of an alkenyl group is independently optionally substituted,i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is unsubstituted C2-10 alkenyl. Incertain embodiments, the alkenyl group is substituted C2-10 alkenyl.

“Alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C2-20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10carbon atoms (“C2-10 alkynyl”). In some embodiments, an alkynyl grouphas 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, analkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In someembodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”).In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms(“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynylgroup has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of C2-4 alkynyl groups include, withoutlimitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl(C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groupsinclude the aforementioned C2-4 alkynyl groups as well as pentynyl (C5),hexynyl (C6), and the like. Additional examples of alkynyl includeheptynyl (C7), octynyl (C8), and the like. Unless otherwise specified,each instance of an alkynyl group is independently optionallysubstituted, i.e., unsubstituted (an “unsubstituted alkynyl”) orsubstituted (a “substituted alkynyl”) with one or more substituents. Incertain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl.In certain embodiments, the alkynyl group is substituted C2-10 alkynyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In certain embodiments, the heteroatom is independentlyselected from nitrogen, sulfur, and oxygen. In heterocyclyl groups thatcontain one or more nitrogen atoms, the point of attachment can be acarbon or nitrogen atom, as valency permits. A heterocyclyl group caneither be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic heterocyclyl”),and can be saturated or partially unsaturated. Heterocyclyl bicyclicring systems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclic ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclicring, or ring systems wherein the heterocyclic ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclic ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclic ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms in the aromatic ring system (“C6-14 aryl”). In someembodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g.,phenyl). In some embodiments, an aryl group has ten ring carbon atoms(“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In someembodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”;e.g., anthracyl). “Aryl” also includes ring systems wherein the arylring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Unlessotherwise specified, each instance of an aryl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C6-14 aryl. Incertain embodiments, the aryl group is substituted C6-14 aryl.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, which are divalent bridging groups arefurther referred to using the suffix -ene, e.g., alkylene, alkenylene,alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene.

The term “alkoxy” or “alkyloxy” refers to an —O-alkyl radical, whereinalkyl is optionally substituted alkyl as defined herein. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

The term “aryloxy” refers to an —O-aryl, wherein aryl is optionallysubstituted aryl as defined herein.

As used herein, the term “optionally substituted” refers to asubstituted or unsubstituted moiety.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

“Acyl” as used herein refers to a moiety selected from the groupconsisting of —C(═O)Raa,—CHO, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa,—C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —C(═O)NRbbSO2Raa, —C(═S)N(Rbb)2,—C(═O)SRaa, and —C(═S)SRaa, wherein Raa and Rbb are as defined herein.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substituents include, but are not limitedto, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2,—CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2,—SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc,—C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2,C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 memberedheteroaryl, or two Rcc groups attached to a nitrogen atom are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups, and wherein Raa, Rbb, Rcc, and Rdd are as defined above.

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to, —Raa,—N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa,—C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3,—P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2,—P(═O)2N(Rbb)2, and —P(═0)(NRbb)2, wherein Raa, Rbb, and Rcc are asdefined herein. Oxygen protecting groups are well known in the art andinclude those described in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999,incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9- (9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (ColdSpring Harbor Laboratory Press, 1988); and Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans usually consist solely of O-glycosidic linkagesbetween monosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of β-1,4-linked D-glucose, and chitin isa glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can behomo or heteropolymers of monosaccharide residues, and can be linear orbranched. Glycans can be found attached to proteins as in glycoproteinsand proteoglycans. They are generally found on the exterior surface ofcells. O- and N-linked glycans are very common in eukaryotes but mayalso be found, although less commonly, in prokaryotes. N-Linked glycansare found attached to the R-group nitrogen (N) of asparagine in thesequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is anyamino acid except praline.

As used herein, the term “antigen” is defined as any substance capableof eliciting an immune response.

As used herein, the term “immunogenicity” refers to the ability of animmunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “CD1d” refers to a member of the CD1 (clusterof differentiation 1) family of glycoproteins expressed on the surfaceof various human antigen-presenting cells. CD1d presented lipid antigensactivate natural killer T cells. CD1d has a deep antigen-binding grooveinto which glycolipid antigens bind. CD1d molecules expressed ondendritic cells can bind and present glycolipids, including alpha-GalCeranalogs such as C34.

As used herein, the term “epitope” is defined as the parts of an antigenmolecule which contact the antigen binding site of an antibody or a Tcell receptor.

As used herein, the term “vaccine” refers to a preparation that containsan antigen, consisting of whole disease-causing organisms (killed orweakened) or components of such organisms, such as proteins, peptides,or polysaccharides, that is used to confer immunity against the diseasethat the organisms cause. Vaccine preparations can be natural, syntheticor derived by recombinant DNA technology.

As used herein, the term “antigen specific” refers to a property of acell population such that supply of a particular antigen, or a fragmentof the antigen, results in specific cell proliferation.

As used herein, the term “specifically binding,” refers to theinteraction between binding pairs (e.g., an antibody and an antigen). Invarious instances, specifically binding can be embodied by an affinityconstant of about 10-6 moles/liter, about 10-7 moles/liter, or about10-8 moles/liter, or less.

As used herein, the terms glycoenzymes refers to at least in part theenzymes in the globo-series biosynthetic pathway; exemplary glycoenzymesinclude alpha-4GalT; beta-4GalNAcT-I; or beta-3GalT-V enzymes.

As used herein, the term “globo-series pathway” includes to abiosynthetic and enzymatic pathways described in FIG. 1.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In one embodiment, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and in some embodiments more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with anantibody of the invention. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include cancer.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, leukemia and other lymphoproliferative disorders, andvarious types of head and neck cancer.

The term “globo-series -related disorder” refers to or describes adisorder that is typically characterized by or contributed to byaberrant functioning or presentation of the pathway. Examples of suchdisorders include, but are not limited to, hyperproliferative diseases,including cancer.

Examples of immunologic deficiency syndromes include, but are notlimited to, ataxia telangiectasia, leukocyte-adhesion deficiencysyndrome, lymphopenia, dysgammaglobulinemia, HIV or deltaretrovirusinfections, common variable immunodeficiency, severe combinedimmunodeficiency, phagocyte bactericidal dysfunction,agammaglobulinemia, DiGeorge syndrome, and Wiskott-Aldrich syndrome.Examples of hypersensitivity include, but are not limited to, allergies,asthma, dermatitis, hives, anaphylaxis, Wissler's syndrome, andthrombocytopenic purpura.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing or decreasing inflammation and/or tissue/organdamage, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

An “individual” or a “subject” is a vertebrate. In certain embodiments,the vertebrate is a mammal. Mammals include, but are not limited to,farm animals (such as cows), sport animals, pets (such as cats, dogs,and horses), primates, mice and rats. In certain embodiments, thevertebrate is a human.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. In certainembodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the substance/molecule are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At211, 1131, 1125, Y90, Rel86, Rel88, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolyticenzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegaIl (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

In one aspect, the present disclosure is based on the surprisingdiscovery that the modification of the stage-specific embryonic antigens(SSEA3 and SSEA4) with certain groups elicited robust IgG antibodyresponse to specifically recognize SSEA3 and SSEA4, respectively.

In some examples, the modification of SSEA3 comprises a fluoro, an azidoor an O-phenyl group at the one or more positions of the glucose ofSSEA3. In some examples, the modification of SSEA3 comprises a fluoro,an azido or an O-phenyl group at the one or more positions of thenon-reducing end galactose. In some examples, the modification of SSEA4comprises a fluoro, an azido or an O-phenyl group at one or morepositions of the glucose of SSEA4. In some examples, the modification ofSSEA4 comprises a fluoro, an azido or an O-phenyl group at one or morepositions of the sialic acid residue.

In certain aspects, the present disclosure provides SSEA3 and SSEA4analogs having the modification at the reducing and/or non-reducing end.Such SSEA3 and SSEA4 analogs can elicit a stronger immune response(e.g., induction of IgG antibodies against SSEA3 and/or SSEA4) ascompared to the native SSEA3 and SSEA4. The antibodies induced by animmunogenic composition comprising such unnatural glycan moiety are ableto mediate the complement-dependent cell cytotoxicity against tumorcells.

Compounds

Accordingly, the present invention also features novel compoundsconsisting of the modified carbohydrate antigens (SSEA3 and SSEA4),glycan conjugates comprising such, and immunogenic compositions andvaccines thereof

In one aspect, the present invention provides a compound of formula (I):

or a salt thereof,

wherein:

-   -   X1 is —OR or —SR, wherein R is hydrogen, a oxygen or sulfur        protecting group, optionally substituted C1-10 alkyl, optionally        substituted aryl, optionally substituted acyl, or optionally        substituted imidoyl;    -   each instance of R1, R2, R3, R4, R5, R6 and L is independently        selected from hydrogen, halogen, optionally substituted alkyl,        optionally substituted alkenyl, optionally substituted alkynyl,        optionally substituted heterocyclyl, optionally substituted        aryl, —N3, —NO2, —N(RB)2, —N(RA)C(O)RA, —ORA, —OC(O)RA, —SRA,        —C(O)N(RB)2, —CN, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA,        —SO2N(RB)2, and —NHSO2RB;    -   each instance of RA is independently selected from hydrogen,        optionally substituted alkyl, optionally substituted alkenyl,        optionally substituted alkynyl, optionally substituted        heterocyclyl, and optionally substituted aryl;    -   each instance of RB is independently selected from hydrogen,        optionally substituted alkyl, optionally substituted alkenyl,        optionally substituted alkynyl, optionally substituted        heterocyclyl, and optionally substituted aryl; and    -   provided the compound is not of the formula:

In certain embodiments, X1 is in the alpha configuration. In certainembodiments, X1 is in the beta configuration.

In some embodiments, X1 is —ORA. In some embodiments, X1 is —OH. In someembodiments, X1 is —O(protecting group). In some embodiments, X1 is—ORA, wherein RA is unsubstituted C1-10 alkyl. In some embodiments, X1is —ORA, wherein RA is substituted C1-10 alkyl. In some embodiments, X1is —ORA, wherein RA is unsubstituted aryl. In some embodiments, X1 is—ORA, wherein RA is substituted aryl. In some embodiments, X1 is —ORA,wherein RA is unsubstituted acyl. In some embodiments, X1 is —ORA,wherein RA is substituted acyl. In some embodiments, X1 is —ORA, whereinRA is unsubstituted imidoyl. In some embodiments, X1 is —ORA, wherein RAis substituted imidoyl.

In some embodiments, X1 is —SRA. In some embodiments, X1 is —SH. In someembodiments, X1 is —S(protecting group). In some embodiments, X1 is—SRA, wherein RA is unsubstituted C1-10 alkyl. In some embodiments, X1is —SRA, wherein RA is substituted C1-10 alkyl. In certain embodiments,X1 is —SCH3. In some embodiments, X1 is —SRA, wherein RA isunsubstituted aryl. In some embodiments, X1 is —SRA, wherein RA issubstituted aryl. In some embodiments, X1 is —SRA, wherein RA isunsubstituted acyl. In some embodiments, X1 is —SRA, wherein RA issubstituted acyl. In some embodiments, X1 is —SRA, wherein RA isunsubstituted imidoyl. In some embodiments, X1 is —SRA, wherein RA issubstituted imidoyl.

In some embodiments, X1 is C1-10 alkoxy. In some embodiments, X1 is C1-3alkoxy.

In some embodiments, X1 is selected from the group consisting ofalpha-thiomethyl, beta-thiomethyl, alpha-thiocresyl, beta-thiocresyl,alpha-t-butyldiphenylsilyloxy, beta-t-butyldiphenylsilyloxy, andalpha-methoxy.

In some embodiments, R1 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R1 is —N3. In certain embodiments, R1 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R1 is —NH2. In certain embodiments, R1 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R1 is—N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R1 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2.

In certain embodiments, R1 is —NH(Cbz). In certain embodiments, R1 is—NH(Fmoc). In certain embodiments, R1 is —NHC(O)CCl3. In certainembodiments, R1 is —NHC(O)CH3. In certain embodiments, R1 is—N(C(O)CH3)2.

In some embodiments, R2 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R2 is —N3. In certain embodiments, R2 is —N3 or —N(RW)2,wherein each RW is independently hydrogen or a nitrogen protectinggroup. In certain embodiments, R2 is —NH2. In certain embodiments, R2 is—NHRW, wherein RW is a nitrogen protecting group. In certainembodiments, R2 is —N(RW)2, wherein each RW is a nitrogen protectinggroup. In certain embodiments, R2 is selected from the group consistingof —N3, —NH(Cbz), —NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and—N(C(O)CH3)2. In certain embodiments, R2 is —NH(Cbz). In certainembodiments, R2 is —NH(Fmoc). In certain embodiments, R2 is —NHC(O)CCl3.In certain embodiments, R2 is —NHC(O)CH3. In certain embodiments, R2 is—N(C(O)CH3)2.

In some embodiments, R3 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R3 is —N3. In certain embodiments, R3 is —N3 or —N(RW)2,wherein each RW is independently hydrogen or a nitrogen protectinggroup. In certain embodiments, R3 is —NH2. In certain embodiments, R3 is—NHRW, wherein RW is a nitrogen protecting group. In certainembodiments, R3 is —N(RW)2, wherein each RW is a nitrogen protectinggroup. In certain embodiments, R3 is selected from the group consistingof —N3, —NH(Cbz), —NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and—N(C(O)CH3)2. In certain embodiments, R3 is —NH(Cbz). In certainembodiments, R3 is —NH(Fmoc). In certain embodiments, R3 is —NHC(O)CCl3.In certain embodiments, R3 is —NHC(O)CH3. In certain embodiments, R3 is—N(C(O)CH3)2.

In some embodiments, R4 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R4 is —N3. In certain embodiments, R4 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R4 is —NH2. In certain embodiments, R4 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R4 is—N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R4 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2.

In certain embodiments, R4 is —NH(Cbz). In certain embodiments, R4 is—NH(Fmoc). In certain embodiments, R4 is —NHC(O)CCl3. In certainembodiments, R4 is —NHC(O)CH3. In certain embodiments, R4 is—N(C(O)CH3)2.

In some embodiments, R5 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R5 is —N3. In certain embodiments, R5 is —N3 or —N(RW)2,wherein each RW is independently hydrogen or a nitrogen protectinggroup. In certain embodiments, R5 is —NH2. In certain embodiments, R5 is—NHRW, wherein RW is a nitrogen protecting group. In certainembodiments, R5 is —N(RW)2, wherein each RW is a nitrogen protectinggroup. In certain embodiments, R5 is selected from the group consistingof —N3, —NH(Cbz), —NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and—N(C(O)CH3)2. In certain embodiments, R5 is —NH(Cbz). In certainembodiments, R5 is —NH(Fmoc). In certain embodiments, R5 is —NHC(O)CCl3.In certain embodiments, R5 is —NHC(O)CH3. In certain embodiments, R5 is—N(C(O)CH3)2.

In some embodiments, R6 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R6 is —N3. In certain embodiments, R6 is —N3 or —N(RW)2,wherein each RW is independently hydrogen or a nitrogen protectinggroup. In certain embodiments, R6 is —NH2. In certain embodiments, R6 is—NHRW, wherein RW is a nitrogen protecting group. In certainembodiments, R6 is —N(RW)2, wherein each RW is a nitrogen protectinggroup. In certain embodiments, R6 is selected from the group consistingof —N3, —NH(Cbz), —NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and—N(C(O)CH3)2. In certain embodiments, R6 is —NH(Cbz). In certainembodiments, R6 is —NH(Fmoc). In certain embodiments, R6 is —NHC(O)CCl3.In certain embodiments, R6 is —NHC(O)CH3. In certain embodiments, R6 is—N(C(O)CH3)2.

In some embodiments, R1, R2 and R3 are the same. In some embodiments,R1, R2 and R3 are —OH. In some embodiments, R4, R5 and R6 are the same.In some embodiments, R4, R5 and R6 are —OH.

In certain embodiments, L is —OH.

In certain embodiments, L is —OH and R1 is —N3. In certain embodiments,L is —OH, R1 is —N3, and each instance of R2, R3, R4, R5 and R6 is —OH.

In certain embodiments, L is —OH and R2 is —N3. In certain embodiments,L is —OH, R2 is —N3, and each instance of R1, R3, R4, R5 and R6 is —OH.

In certain embodiments, L is —OH and R3 is —N3. In certain embodiments,L is —OH, R3 is —N3, and each instance of R1, R2, R4, R5 and R6 is —OH.

In certain embodiments, L is —OH and R4 is —N3. In certain embodiments,L is —OH, R4 is —N3, and each instance of R1, R2, R3, R5 and R6 is —OH.

In certain embodiments, L is —OH and R5 is —N3. In certain embodiments,L is —OH, R5 is —N3, and each instance of R1, R2, R3, R4 and R6 is —OH.

In certain embodiments, L is —OH and R6 is —N3. In certain embodiments,L is —OH, R6 is —N3, and each instance of R1, R2, R3, R4 and R5 is —OH.

In certain embodiments, each instance of R1, R2, R3, R4, R5, R6 and L is—F. In certain embodiments, R1 is —F. In certain embodiments, R2 is —F.In certain embodiments, R3 is —F. In certain embodiments, R4 is —F. Incertain embodiments, R5 is —F. In certain embodiments, R6 is —F. Incertain embodiments, L is —F.

In certain embodiments, L is of the following structure:

wherein:

-   -   each instance of R8, R9, R10 and R11 is independently selected        from hydrogen, halogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted heterocyclyl, optionally substituted aryl, —N3,        —NO2, —N(RB)2, —N(RA)C(O)RA, —ORA, —OC(O)RA, —SRA, — C(O)N(RB)2,        —CN, —C(O)RA, —C(O)ORA, —S(O)RA, —SO2RA, —SO2N(RB)2, and        —NHSO2RB;    -   R_(N) is selected from —N3, —NO2, —N(RB)2, —N(RA)C(O)RA, —ORA,        —OC(O)RA, —SRA, —C(O)N(RB)2, —CN, —C(O)RA, —C(O)ORA, —S(O)RA,        —SO2RA, —SO2N(RB)2, and —NHSO2RB;    -   each instance of RA is independently selected from hydrogen,        optionally substituted alkyl, optionally substituted alkenyl,        optionally substituted alkynyl, optionally substituted        heterocyclyl, and optionally substituted aryl; and    -   each instance of RB is independently selected from hydrogen,        optionally substituted alkyl, optionally substituted alkenyl,        optionally substituted alkynyl, optionally substituted        heterocyclyl, and optionally substituted aryl.

In some embodiments, the compound is of Formula (II)

wherein: R1, R2, R3, R8, R9, R10, R11 and R_(N) and X1 are as describedherein, and

provided the compound is not of the formula:

In some embodiments, R8 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R8 is —N3. In certain embodiments, R8 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R8 is —NH2. In certain embodiments, R8 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R8 is—N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R8 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2. Incertain embodiments, R8 is —NH(Cbz). In certain embodiments, R8 is—NH(Fmoc). In certain embodiments, R8 is —NHC(O)CCl3. In certainembodiments, R8 is —NHC(O)CH3. In certain embodiments, R8 is—N(C(O)CH3)2.

In some embodiments, R9 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R9 is —N3. In certain embodiments, R9 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R9 is —NH2. In certain embodiments, R9 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R9 is—N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R9 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2. Incertain embodiments, R9 is —NH(Cbz). In certain embodiments, R9 is—NH(Fmoc). In certain embodiments, R9 is —NHC(O)CCl3. In certainembodiments, R9 is —NHC(O)CH3. In certain embodiments, R9 is—N(C(O)CH3)2.

In some embodiments, R10 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R10 is —N3. In certain embodiments, R10 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R10 is —NH2. In certain embodiments, R10 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R10is —N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R10 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2. Incertain embodiments, R10 is —NH(Cbz). In certain embodiments, R10 is—NH(Fmoc). In certain embodiments, R10 is —NHC(O)CCl3. In certainembodiments, R10 is —NHC(O)CH3. In certain embodiments, R10 is—N(C(O)CH3)2.

In some embodiments, R11 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R11 is —N3. In certain embodiments, R11 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R11 is —NH2. In certain embodiments, R11 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R11is —N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R11 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2. Incertain embodiments, R11 is —NH(Cbz). In certain embodiments, R11 is—NH(Fmoc). In certain embodiments, R11 is —NHC(O)CCl3. In certainembodiments, R11 is —NHC(O)CH3. In certain embodiments, R11 is—N(C(O)CH3)2.

In some embodiments, R12 is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, R12 is —N3. In certain embodiments, R12 is —N(RW)2, whereineach RW is independently hydrogen or a nitrogen protecting group. Incertain embodiments, R12 is —NH2. In certain embodiments, R12 is —NHRW,wherein RW is a nitrogen protecting group. In certain embodiments, R12is —N(RW)2, wherein each RW is a nitrogen protecting group. In certainembodiments, R12 is selected from the group consisting of —N3, —NH(Cbz),—NH(Boc), —NH(Fmoc), —NHC(O)CCl3, —NHC(O)CH3, and —N(C(O)CH3)2. Incertain embodiments, R12 is —NH(Cbz). In certain embodiments, R12 is—NH(Fmoc). In certain embodiments, R12 is —NHC(O)CCl3. In certainembodiments, R12 is —NHC(O)CH3. In certain embodiments, R12 is—N(C(O)CH3)2.

In some embodiments, R_(N) is —N3 or —N(RW)2, wherein each RW isindependently hydrogen or a nitrogen protecting group. In certainembodiments, RN is —N3. In certain embodiments, R_(N) is —N(RW)2,wherein each RW is independently hydrogen or a nitrogen protectinggroup. In certain embodiments, R_(N) is —NH2. In certain embodiments,R_(N) is —NHRW, wherein RW is a nitrogen protecting group. In certainembodiments, R_(N) is —N(RW)2, wherein each RW is a nitrogen protectinggroup. In certain embodiments, R_(N) is selected from the groupconsisting of —N3, —NH(Cbz), —NH(Boc), —NH(Fmoc), —NHC(O)CCl3,—NHC(O)CH3, and —N(C(O)CH3)2. In certain embodiments, R_(N) is —NH(Cbz).In certain embodiments, R_(N) is —NH(Fmoc). In certain embodiments,R_(N) is —NHC(O)CCl3. In certain embodiments, R_(N) is —NHC(O)CH3. Incertain embodiments, R_(N) is —N(C(O)CH3)2.

Immunogenic Compositions

In another aspect, the present invention provides an immunogeniccomposition, comprising (a) a glycan conjugate including a carrier andone or more glycans, and optionally (b) an adjuvant,

wherein: each of the one or more glycans is conjugated with the carrierthrough a linker, having the formula (III) or (IV):

wherein X1, R1, R2, R3, R4, R5, R6, R8, R9, R10, R11, L and R_(N) are asdescribed herein.

In certain embodiments, the linker is a hetero- or homo-bifunctionallinker.

In certain embodiments, the linker is a homo-bifunctional p-nitrophenyllinker.

In certain embodiments, the linker includes at least one sulfur atom,carboxylate group, amide group, carbamate group, carbonate group,thiocarbamate group, thiocarbonate group, thioether group, succinamidegroup, n-hydroxy succinamide group, or any combination thereof.

In certain embodiments, the linker is wherein —L¹—L²— is a bond, O, S ,NRL1a—, —C(═O)—, —NRL1 aC(═O)—, —NRL1 aC(═O)O—, —C(═O)NRL1a—,—OC(═O)NRL1a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL1aC(═S)—,—C(═S)NRL1a—, trans-CRL1b=CRL1b—, cis-CRL1b=CRL1b , C≡C , OC(RL1b)2-,—C(RL1b)2O—, —NRL1aC(RL1b)2—, —C(RL1b)2NRL 1 a—, —SC(RL1b)2-,—C(RL1b)2S—, —S(═O)2O—, —OS(═O)2—, —S(═O)2NRL1a—, —NRL1aS(═O)2-, or anoptionally substituted C1-20 hydrocarbon chain, optionally wherein oneor more carbon units of the hydrocarbon chain is replaced with —O—, —S—,—NRL1a—, —C(═O)—, NRL1 aC(═O)—, —NRL1 aC(═O)O—, —C(═O)NRL 1 a—,—OC(═O)NRL 1 a—, —S C(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NRL1aC(═S)—,—C(═S)NRL1a—, trans-CRL1b=CRL1b—, cis-CRL1b=CRL1b , C≡C , S(═O)20-,—OS(═O)2—, —S(═O)2NRL1a—, or —NRL1aS(═O)2—, wherein RL1a is hydrogen,optionally substituted C1-6 alkyl, or a nitrogen protecting group, orRL1a is joined with the adjacent carbon atom to form an optionallysubstituted heterocyclic ring, and wherein each occurrence of RL1b isindependently selected from the group consisting of hydrogen, halogen,optionally substituted C1-10 alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, andoptionally substituted heteroaryl, or RL1b is joined with the adjacentcarbon or nitrogen or oxygen atom to form an optionally substitutedcarbocyclic or heterocyclic ring, or two RL1b groups are joined to forman optionally substituted carbocyclic or optionally substitutedheterocyclic ring; and L2 is a moiety derived from a crosslinkingreagent capable of crosslinking the carrier and L1.

The carrier can be a protein, a lipid, a lipolized protein, a virus, apeptide, or a dendrimer of glycopeptides. In certain embodiments, thecarrier is a peptide comprising a T cell epitope.

Examples of carrier proteins which may be used in the present inventionare tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria toxincross-reacting material 197 (CRM197), fragment C of TT, Keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), protein D, outer-membraneprotein (OMP) and pneumolysin, diphtheria toxin cross-reacting material197 (CRM197) or other DT point mutants, such as CRM176, CRM228, CRM 45(Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45,CRM102, CRM 103 and CRM107 and other mutations described in the art.

In certain embodiments, the glycan conjugate is of the formula (IV-a) or(IV-b):

wherein m is an integer of 1 to 40, inclusive.

In certain embodiments, m is an integer of 1 to 30, inclusive. Asgenerally defined herein, m is an integer of 1 to 20 inclusive. Incertain embodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 4. In certain embodiments, m is 6. In certainembodiments, m is 8. In certain embodiments, m is 10. In certainembodiments, m is 15. In certain embodiments, m is 20. In certainembodiments, m is 30. In certain embodiments, m is 40.

In another aspect, the present invention provides a glycan conjugatemixture comprising at least two of the glycan conjugates as describedherein. In certain embodiments, the average value of w in the glycanmixture is from about 1.0 to about 40.0. In certain embodiments, theaverage value of w in the glycan mixture is from about 1.0 to 10.0. Incertain embodiments, the average value of w in the glycan mixture isabout 5.7, 4.9, 2.9, 2.8, or 3.1. In certain embodiments, the averagevalue of w in the glycan mixture is about 4.9, 2.9, 2.8, or 3.1.

In certain embodiments, the immunogenic compositions described hereininclude an immunogenically effective amount of a glycan conjugate of theinvention. In certain embodiments, the immunogenic composition includesa pharmaceutically effective amount of the inventive glycan conjugate.

The compounds of the invention can be synthesized using proceduresdescribed herein and also see US20140051127.

The immunogenic conjugate of the invention may include one or moremolecules (e.g., 1-40, 1-20, 1-25, 1-30,) of the same or different SSEA3and/or SSEA4 analogs and/or related derivatives. Additional descriptionsand related procedures for generating glycan conjugates are describedbelow. Also see U.S. Pat. No. 8,268,969. The contents of which is herebyincorporated by reference.

In certain embodiments, the immunogenic composition of the invention mayinclude one or more adjuvants. Suitable adjuvants can include, forexample, C34, 7DW8-5, C17, C23, C-30, alpha-galactoceramide, Gluco-C34,Aluminum salt, Squalene, MF59, and QS-21).

As used herein, the term “alum adjuvant” refers to an aluminum salt withimmune adjuvant activity. This agent adsorbs and precipitates proteinantigens in solution; the resulting precipitate improves vaccineimmunogenicity by facilitating the slow release of antigen from thevaccine depot formed at the site of inoculation.

As used herein, the term “immunologic adjuvant” refers to a substanceused in conjunction with an immunogen which enhances or modifies theimmune response to the immunogen. The α-GalCer analogs of the presentdisclosure are used as immunologic adjuvants to modify or augment theeffects of a vaccine by stimulating the immune system of a patient whois administered the vaccine to respond to the vaccine more vigorously.In an exemplary implementation, the analog C34 is used as an adjuvant.The structures of C34 and other alpha-galactosyl ceramide analogs andtheir use as adjuvants are disclosed in detail in U.S. Pat. No.7,928,077.

As used herein, the term “glycolipid” refers to a carbohydrate-attachedlipid that serves as a marker for cellular recognition.

The glycolipids C34, Gluco-C34, C23 and 7DW8-5 have the followingstructures:

The immunogenic composition can further include a pharmaceuticallyacceptable excipient. In certain embodiments, the immunogeniccompositions described herein include a pharmaceutically effectiveamount of a glycan conjugate of the invention.

In another aspect, the present invention provides a cancer vaccinecomprising an immunogenic composition described herein and apharmaceutically acceptable excipient.

The cancer vaccines of the invention may include a single dose ormultiple doses of the inventive glycan conjugates, a glycan conjugatemixture thereof, or immunogenic compositions thereof. The providedcancer vaccines may be useful for treating or reducing the risk ofcancers. The cancer vaccines may also include packaging informationdescribing the use or prescribing information for the subject or ahealth care professional. Such information may be required by aregulatory agency such as the U.S. Food and Drug Administration (FDA).The cancer vaccine may also optionally include a device foradministration of the compound or composition, for example, a syringefor parenteral administration.

Pharmaceutical Formulations

The immune composition is administered in a manner compatible with thedosage formulation, and in an amount that is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and variesaccording to the size of the host.

The immune composition of this invention can also be used to generateantibodies in animals for production of antibodies, which can be used inboth cancer treatment and diagnosis. Methods of making monoclonal andpolyclonal antibodies and fragments thereof in animals (e.g., mouse,rabbit, goat, sheep, or horse) are well known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. The term “antibody” includes intactimmunoglobulin molecules as well as fragments thereof, such as Fab,F(ab′)2, Fv, scFv (single chain antibody), and dAb (domain antibody;Ward, et. al. (1989) Nature, 341, 544).

The compositions disclosed herein can be included in a pharmaceuticalcomposition together with additional active agents, carriers, vehicles,excipients, or auxiliary agents identifiable by a person skilled in theart upon reading of the present disclosure.

The pharmaceutical compositions preferably comprise at least onepharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the compositions disclosed herein form the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Clinical Applications

The present invention provides glycan conjugates, immunogeniccompositions or vaccines useful for the treatment of a proliferativedisease such as cancer (e.g. lung cancer, large bowel cancer, pancreascancer, biliary tract cancer, or endometrial cancer), benign neoplasm,or angiogenesis in a subject.

The immunogenic compositions or vaccines described herein can also beused to generate antibodies in human or animals for production ofantibodies, which can be used in both cancer treatment and diagnosis. Insome embodiments, the immunogenic compositions or vaccines describedherein can also be used to generate the production of GloboH, SSEA3and/or SSEA4 antibodies. Methods of making monoclonal and polyclonalantibodies and fragments thereof in human and/or animals (e.g., mouse,rabbit, goat, sheep, or horse) are well known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. The term “antibody” includes intactimmunoglobulin molecules as well as fragments thereof, such as Fab,F(ab′).sub.2, Fv, scFv (single chain antibody), and dAb (domainantibody; Ward, et. al. (1989) Nature, 341, 544).

Compositions comprising at least one anti-SSEA3/SSEA4/GloboH antibody orat least one polynucleotide comprising sequences encoding ananti-SSEA3/SSEA4/GloboH antibody are provided. In certain embodiments, acomposition may be a pharmaceutical composition. As used herein,compositions comprise one or more antibodies that bind to one or moreSSEA3/SSEA4/GloboH and/or one or more polynucleotides comprisingsequences encoding one or more antibodies that bind to one or moreSSEA3/SSEA4/GloboH. These compositions may further comprise suitablecarriers, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art.

Isolated antibodies and polynucleotides are also provided. In certainembodiments, the isolated antibodies and polynucleotides aresubstantially pure.

In one embodiment, anti-SSEA3/SSEA4/GloboH antibodies are monoclonal. Inanother embodiment, fragments of the anti-SSEA3/SSEA4/GloboH antibodies(e.g., Fab, Fab′-SH and F(ab!)2 fragments) are provided. These antibodyfragments can be created by traditional means, such as enzymaticdigestion, or may be generated by recombinant techniques. Such antibodyfragments may be chimeric, humanized, or human. These fragments areuseful for the diagnostic and therapeutic purposes set forth below.

Pharmaceutical Formulations

Therapeutic formulations comprising an pharmaceutical agents of theinvention are prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,histidine and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, including, butnot limited to those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thiol-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Pharmaceutical compositions of the invention can be used to treat,inhibit, delay progression of, prevent/delay recurrence of, ameliorate,or prevent diseases, disorders or conditions associated with abnormalexpression and/or activity of SSEA3/SSEA4/GloboHs and SSEA3/SSEA4/GloboHrelated proteins, including but not limited to cancer, musculardisorders, ubiquitin-pathway-related genetic disorders,immune/inflammatory disorders, neurological disorders, and otherubiquitin pathway-related disorders.

In one aspect, a blocking antibody of the invention is specific for aSSEA3/SSEA4/GloboH.

Pharmaceutical compositions of the invention can be used either alone orin combination with other compositions in a therapy. For instance, anantibody of the invention may be co-administered with another antibody,and/or adjuvant/therapeutic agents (e.g., steroids). For instance, anantibody of the invention may be combined with an anti-inflammatoryand/or antiseptic in a treatment scheme, e.g. in treating any of thediseases described herein, including cancer, muscular disorders,ubiquitin-pathway-related genetic disorders, immune/inflammatorydisorders, neurological disorders, and other ubiquitin pathway-relateddisorders. Such combined therapies noted above include combinedadministration (where the two or more agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,and/or following, administration of the adjunct therapy or therapies.

Pharmaceutical compositions of the invention (and adjunct therapeuticagent) can be administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, thePharmaceutical composition can be suitably administered by pulseinfusion, particularly with declining doses of the antibody. Dosing canbe by any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. Intracellular expression of anintrabody is effected by introducing a nucleic acid encoding the desiredantibody or antigen-binding portion thereof (lacking the wild-typeleader sequence and secretory signals normally associated with the geneencoding that antibody or antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest.

Pharmaceutical compositions of the invention would be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The antibody need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of antibodies of the invention present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anPharmaceutical compositions of the invention (when used alone or incombination with other agents such as chemotherapeutic agents) willdepend on the type of disease to be treated, the type of antibody, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1mg/kg-10 mg/kg) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) maybe administered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the antibody.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice. Moreover,the article of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody of the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

In some embodiments, the provided glycan conjugates, immunogeniccompositions or vaccines are useful in treating, or diagnosing a cancer,including, but are not limited to, acoustic neuroma, adenocarcinoma,adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM),a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronicmyelocytic leukemia (CML), chronic neutrophilic leukemia (CNL),hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g.,neurofibromatosis (NF) type 1 or type 2, schwannomatosis),neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrinetumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g.,cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g.,pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm(IPMN), islet cell tumors), penile cancer (e.g., Paget's disease of thepenis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT),prostate cancer (e.g., prostate adenocarcinoma), rectal cancer,rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamouscell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cellcarcinoma (BCC)), small bowel cancer (e.g., appendix cancer), softtissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma,malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma,fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat glandcarcinoma, synovioma, testicular cancer (e.g., seminoma, testicularembryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of thethyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer),urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's diseaseof the vulva). In certain embodiments, the provided glycan conjugates,immunogenic compositions or vaccines are useful for treating braincancer, lung cancer, breast cancer, oral cancer, esophagus cancer,stomach cancer, liver cancer, bile duct cancer, pancreas cancer, coloncancer, kidney cancer, bone cancer, skin cancer, cervix cancer, ovarycancer, and prostate cancer.

To perform the treatment methods described herein, an effective amountof any of the glycan conjugates or immunogenic compositions or vaccinesdescribed herein may be administered to a subject in need of thetreatment via a suitable route, as described above. The subject, such asa human subject, can be a patient having cancer, suspected of havingcancer, or susceptible to cancer. The amount of the glycan conjugate orimmunogenic composition administered to the subject may be effective ineliciting immune responses specific to the glycan moiety in theconjugate or composition. In some embodiments, the amount of the glycanconjugate or immunogenic composition is sufficient to elicit immuneresponses leading to the inhibition of cancer growth and/or reduction oftumor mass. In other embodiments, the amount of the glycan conjugate orimmunogenic composition may be effective in delaying the onset of thetarget cancer or reducing the risk for developing the cancer. The exactamount of the provided glycan conjugates, immunogenic compositions orvaccines required to achieve an effective amount will vary from subjectto subject, depending, for example, on species, age, and generalcondition of a subject, severity of the side effects or disorder,identity of the particular compound(s), mode of administration, and thelike. The desired dosage can be delivered three times a day, two times aday, once a day, every other day, every third day, every week, every twoweeks, every three weeks, or every four weeks. In certain embodiments,the desired dosage can be delivered using multiple administrations(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, or more administrations).

In certain embodiments, an effective amount of the provided glycanconjugates, immunogenic compositions or vaccines for administration oneor more times a day to a 70 kg adult human may comprise about 0.0001 mgto about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg toabout 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg,about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100mg to about 1000 mg, of a compound per unit dosage form.

In certain embodiments, the provided glycan conjugates, immunogeniccompositions or vaccines may be administered orally or parenterally atdosage levels sufficient to deliver from about 0.001 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 10 mg/kg, and more preferably from about 1 mg/kg to about 25mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic effect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of the provided glycan conjugates,immunogenic compositions or vaccines to an adult. The amount to beadministered to, for example, a child or an adolescent can be determinedby a medical practitioner or person skilled in the art and can be loweror the same as that administered to an adult.

It will be also appreciated that the provided glycan conjugates,immunogenic compositions or vaccines can be administered in combinationwith one or more additional therapeutically active agents. The providedglycan conjugates, immunogenic compositions or vaccines can beadministered in combination with additional therapeutically activeagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body. It will also be appreciated that the therapy employedmay achieve a desired effect for the same disorder, and/or it mayachieve different effects.

The provided glycan conjugates, immunogenic compositions or vaccines canbe administered concurrently with, prior to, or subsequent to, one ormore additional therapeutically active agents. In general, each agentwill be administered at a dose and/or on a time schedule determined forthat agent. In will further be appreciated that the additionaltherapeutically active agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the inventive compoundwith the additional therapeutically active agent and/or the desiredtherapeutic effect to be achieved. In general, it is expected thatadditional therapeutically active agents utilized in combination beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

In certain embodiments, the provided glycan conjugate, immunogeniccomposition or vaccine is administered in combination with one or moreadditional pharmaceutical agents described herein. In certainembodiments, the additional pharmaceutical agent is an anti-canceragent. Anti-cancer agents encompass biotherapeutic anti-cancer agents aswell as chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon a,interferon y), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. Herceptin (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), Vectibix (panitumumab), Rittman (rituximab), Bexxar(tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (Abraxane),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca2+ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765,AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOKTM),SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470,BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036,BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184,XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib(Velcade)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779),everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055(AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (SanofiAventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) andOSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin,pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

In certain embodiments, the subject being treated is a mammal. Incertain embodiments, the subject is a human. In certain embodiments, thesubject is a domesticated animal, such as a dog, cat, cow, pig, horse,sheep, or goat. In certain embodiments, the subject is a companionanimal such as a dog or cat. In certain embodiments, the subject is alivestock animal such as a cow, pig, horse, sheep, or goat. In certainembodiments, the subject is a zoo animal. In another embodiment, thesubject is a research animal such as a rodent, dog, or non-humanprimate. In certain embodiments, the subject is a non-human transgenicanimal such as a transgenic mouse or transgenic pig.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Syntheses of SSEA3 Analogues

A: Chemo-enzymatic synthesis of SSEA3 analog-NH2

The combined compounds Gb4 analog, ATP, UTP, galactose analog,phosphoenolpyruvate, MgCl₂ with enzymes galactokinase (GalK), UDP-sugarpyrophosphorylase (AtUSP), beta-1,3-galactosyltransferase (LgtD,),pyruvate kinase (PK), and inorganic pyrophosphatase (PPA) in thesolution, and the reaction was initiated at room temperature with the pHcontrolled at 7.0, and the reaction was monitored by TLC until no moreproduct could be observed. After completion of the reaction, theproteins in the reaction mixture were removed by heating for 30 minfollowed by centrifugation and filtration with 0.22 □M filter. Thefiltrate was then purified by C-18 gel chromatography. Fractions werecollected and monitored by TLC.

Example 2 Exemplary Syntheses of SSEA4 Analogues

A : Chemical Synthesis of SSEA4-Gc-NH2

Powdered molecular sieves (4 A, 0.5 g) was added to a solution ofacceptor 3 (93 mg, 0.045 mmol) and imidates 6 (76 mg, 0.068 mmol) in 6mL of dichloromethane (CH2C12). The mixture was stirred at roomtemperature for 2 hrs. After cooled to −10° C., TMSOTf (5 μL, 0.03 mmol)was added, and the mixture was stirred at 5° C. (cold room) overnight.The reaction mixture was quenched by the addition of triethylamine (0.5mL), diluted with CH₂Cl₂ and filtered through a pad of celite. Thefiltrate was washed with saturated sodium bicarbonate (NaHCO₃) aqueoussolution, dried over sodium sulfate (Na₂SO₄), filtered, andconcentrated. The residue was purified by flash silica gelchromatography(50-100% EtOAc in Hexane) to afford hexasaccharide 7 contaminated withimpurities from disaccharide imidates 6. The yield was determined by NMR(90 mg, 68%).

Zinc dust (1g) was added to a solution of hexasaccharide 7 (90 mg, 0.03mmol) in glacial acetic acid (5.0 mL) and the mixture was stirred for1-2 hrs, until compound 7 was consumed by TLC analysis. The reactionmixture was diluted with CH2C12, filtered through a pad of celite, andconcentrated under reduced pressure. The residue was dissolved inpyridine/Ac₂O (1:1, 2.0 mL), stirred for 1 h, and concentrated. Theresidue was purified by flash silica get chromatography. The acylatedmaterial was dissolved in anhydrous CH₂Cl₂ and MeOH (2:8, 10 mL) andtreated with NaOMe (45 mg). After stirring at room temperature for 4hrs, water (0.2 mL) was added, and the resulting mixture was stirred for16 hrs. The reaction mixture was neutralized with amberlyst IR-120,filtered, and concentrated. The residue was purified by reverse phasechromatography (RP-18).

Palladium hydroxide (20% in Charcoal, 50 mg) was added to the adduct ina mixture of methanol/water/ Acetic acid (10:10:0.5, 6 mL) and thereaction mixture was stirred at room temperature under a positivepressure of hydrogen for 16 hrs. The reaction mixture was filteredthrough a pad of celite and concentrated. The residue was purified byreverse phase chromatography to afford 8 (17 mg, 43%).

B: Chemoenzymatic Synthesis of SSEA4 analog-NH2

SSEA4 analogs-NH2 were synthesized via enzymatic regeneration strategyas described in Scheme 3. In this system, ManNAc derivatives werereacted with pyruvate and transformed into Neu5Ac analogs by aldolasecatalysis, followed by incorporation with Gb5-NH2 in the regenerationsystem (J. Am. Chem. Soc. 2013, 135, 14831-14839) to obtain theexemplary SSEA4 analogs-NH2.

Detail of the reaction condition is described as follows: Gb5-NH2 (18μmol), CTP (5 μmol), ManNAc derivative (27 μmol), sodium pyruvate (81μmol), PEP (55 μmol), and ATP (5 μmol), were dissolved in 50 mM Tris-HClbuffer (pH 8.0). Enzymes alpha-(2,3)-sialyltransferase (20 units),sialic acid aldolase(20 units) CMK (10 units), Pykf (10 units), PPA (10units), and Pmcss (10 units) were added to the solution, and thereaction was incubated at 37° C. for 8 hours and monitored by TLC plate.At the end of reaction, enzyme was denatured by heating at 100° C. for 5minutes. The desired SSEA4 analog-NH2 was purified by G25, DEAE, and SPcolumn (80%).

¹H NMR of SSEA4analogs-NH2

B-1. SSEA4-pentylamine (RN=NHAc, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.94 (d, J=3.8 Hz, 1H), 4.72 (d, J=8.5 Hz, 1H),4.54-4.50 (m, 3H), 4.40 (t, J=6.4 Hz, 1H), 4.27 (d, J=2.0 Hz, 1H), 4.20(d, J=2.8 Hz, 1H), 4.10-3.54 (m, 37 H), 3.34-3.31 (m, 1H), 3.02 (t,J=7.6 Hz, 2H), 2.78 (dd, J=12.4, 4.6 Hz, 1H), 2.05 (m, 6H), 1.80 (t,12.2 Hz, 1H), 1.74-1.67 (m, 4H), 1.51-1.45 (m, 2H)

B-2. Neu5Gc_SSEA4-pentylamine (RN=NHGc, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.89 (d, J=3.6 Hz, 1H), 4.66 (d, J=8.2 Hz, 1H),4.52-4.45 (m, 3H), 4.37 (t, J=6.8 Hz, 1H), 4.23 (d, J=3.2 Hz, 1H), 4.15(d, J=2.8 Hz, 1H), 4.10-3.48 (m, 35 H), 3.27 (m, 1H), 2.98 (t, J=7.6 Hz,2H), 2.73 (dd, J=4.8, 12.4 Hz, 1H), 2.00 (s, 3H), 1.77 (t, J=12.0 Hz,1H), 1.72-1.61 (m, 4H), 1.48-1.39 (m, 2 H).

B-3. Ac-Alkynyl_SSEA4-pentylamine (RN=NHCOC₂H₄C≡CH, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.89 (d, J=4.0 Hz, 1H), 4.67 (d, J=8.4 Hz, 1H),4.52-4.45 (m, 3H), 4.37 (t, J=6.4 Hz, 1H), 4.23 (d, J=2.4 Hz, 1H),4.08-3.54 (m, 38 H), 3.28 (m, 1H), 2.99 (t, J=7.6 Hz, 2H), 2.53-2.4 (m,4H), 2.37 (s, 1H), 2.01 (s, 3H), 1.77 (t, J=12.0 Hz, 1H), 1.72-1.62 (m,4H), 1.49-1.41 (m, 2 H).

B-4. Ac-Fluoride_SSEA4-pentylamine (RN=NHCOCH₂F, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.90 (d, J=46.4 Hz, 2H), 4.90 (d, J=4.0 Hz, 1H), 4.67 (d, J=8.8 Hz, 1H), 4.53-4.46 (m, 3H), 4.37 (t, J=6.8 Hz, 1 H),4.24 (d, J=2.8 Hz, 2H), 4.16 (d, J=3.2 Hz, 1 H), 4.09-3.51 (m, 34H),3.28 (m, 1H), 2.99 (t, J=7.2 Hz, 1H), 2.75 (dd, J=4.8, 12.4 Hz, 1H),2.01 (s, 3H), 1.79 (t, J=12.0 Hz, 1H), 1.72-1.62 (m, 4H), 1.48-1.40 (m,2 H).

B-5. Ac-Phenyl _SSEA4-pentylamine (RN=NHCOCH₂Ph, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.39-7.30 (m, 5H), 4.90 (d, J=4.0 Hz, 1H), 4.66(d, J=8.4 Hz, 1H), 4.52-4.46 (m, 3H), 4.37 (t, J=6.8 Hz, 1H), 4.23 (d,J=2.8 Hz, 1H), 4.15 (d, J=3.2 Hz, 1H), 4.08-3.47 (m, 38H), 3.36(dd,J=1.6, 9.2 Hz, 1H), 3.28 (m, 1H), 2.99 (t, J=7.6 Hz, 2H), 2.73 (dd,J=4.8, 12.4 Hz, 1H), 2.00 (s, 3H), 1.76 (t, J=12.0 Hz, 1H), 1.72-1.61(m, 4H), 1.51-1.40 (m, 2 H).

B-6. Ac-Azido_SSEA4-pentylamine (RN=NHCOCH₂N₃, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.88 (d, J=3.6 Hz, 1H), 4.66 (d, J=8.4 Hz, 1H),4.52-4.44 (m, 3H), 4.36 (t, J=6.4 Hz, 1H), 4.23 (d, J=2.4 Hz, 1H),4.08-3.54 (m, 35 H), 3.27 (m, 1H), 2.98 (t, J=7.2 Hz, 2H), 2.73 (dd,J=4.8, 12.4 Hz, 1H), 2.00 (s, 3H), 1.77 (t, J=12.4 Hz, 1H), 1.72-1.60(m, 4H), 1.48-1.39 (m, 2 H).

B-7. 5′-Azido_SSEA4-pentylamine (RN=N₃, R10=OH)

¹H NMR (400 MHz, D₂O): δ 4.90 (d, J=3.6 Hz, 1H), 4.67 (d, J=8.4 Hz, 1H),4.51-4.47 (m, 3H), 4.37 (t, J=6.4 Hz, 1H), 4.23 (d, J=2.8 Hz, 1H), 4.15(d, J=3.2 Hz, 1H), 4.08-3.44 (m, 35H), 3.31-3.27 (m, 1H), 2.99 (t, J=7.2Hz, 1H), 2.73 (dd, J=4.8, 12.4 Hz, 1H), 2.01 (s, 3H), 1.76 (t, J=12.0Hz, 1H), 1.72-1.63 (3, 4H), 1.48-1.41 (m, 2H); HRMS (ESI-TOF, M-H-)C₄₆H₇₈N₅O₃₃- calcd for 1228.4579, found 1228.4621.

B-8. 9′-Azido_SSEA4-pentylamine (RN=NHAc, R10=N₃)

¹H NMR (400 MHz, D₂O) δ 4.85 (d, J=3.8 Hz, 1H), 4.67 (d, J=8.4 Hz, 1H),4.51-4.44 (m, 3H), 4.37 (t, J=6.4 Hz, 1H), 4.23 (d, J=2.8 Hz, 1H),4.10-3.40 (m, 33 H), 3.27 (m, 1H), 2.98 (t, J=7.6 Hz, 2H), 2.72 (dd,J=4.8, 12.8 Hz, 1H), 2.00 (s, 3H), 2.00 (s, 3H), 1.75 (t, J=12.4 Hz,1H), 1.72-1.60 (m, 4H), 1.58-1.38 (m, 2 H).

B-9. NHBz_SSEA4-pentylamine (RN=NHBz, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.80-7.73 (m, 2H), 7.63 (m, 1H), 7.56-7.51 (m,2H), 4.92 (d, J=4.0 Hz, 1H), 4.70 (d, J=8.4 Hz, 1H), 4.58-4.47 (m, 3H),4.40 (t, J=6.4 Hz, 1H), 4.26 (d, J=2.8 Hz, 1H), 4.19 (d, J=3.2 Hz, 1H),4.15-3.53 (m, 36H), 3.31 (m, 1H), 3.01 (t, J=7.6 Hz, 2H), 2.82 (dd,J=4.4, 12.4 Hz, 1H), 2.00 (s, 3H), 1.87 (t, J=12.0 Hz, 1H), 1.72-1.60(m, 4H), 1.48-1.39 (m, 2 H).

C: Cross-Linking Reaction for SSEA4 Analog-SH

In certain embodiments, DTSSP (2.0 eq) and SSEA4 analog-NH2 (1.0 eq) wasmixed in 0.1 M phosphate buffer, pH 7.4 (˜3 mg/ml). The solution wasstirred at room temperature for overnight. Then the reaction mixture waswarmed to 40° C. and added with DTT (9.0 eq). After stirring for 1.5 hrsat 40° C., the reaction mixture was concentrated in vacuo, and theresidue was purified by LH-20 column to afford a white solid SSEA4analogs-SH. (Scheme 4)

¹H NMR of SSEA4 analogs-SH

C-1: SSEA4-SH (RN=NHAc, R10=OH)

¹H NMR (400 Hz, D₂O) δ 4.88 (d, J=4.0 Hz, 1H), 4.65 (d, J=8.5 Hz, 1H),4.50-4.44 (m, 3H), 4.36 (t, J=6.5 Hz, 1H), 4.22 (d, J=2.9 Hz, 1H), 4.14(d, J=3.1 Hz, 1H), 4.04-3.55 (m, 35H), 3.26 (t, J=8.5 Hz, 1H), 3.18 (t,J=6.8 Hz, 2H), 2.74-2.70 (m, 3H), 2.49 (t, J=6.8 Hz, 2H), 1.994 (s, 3H),1.992 (s, 3H), 1.75 (t, J=12.2 Hz, 1H), 1.61 (tt, J=6.7, 6.7 HZ, 2H),1.52 (tt, J=7.1, 7.1 Hz, 2H), 1.40-1.36 (m, 2H);

C-2: Neu5Gc_SSEA4-SH (RN=NHGc, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.89 (d, J=3.9 Hz, 1H), 4.66 (d, J=8.5 Hz, 1H),4.53- 4.43 (m, 3H), 4.36 (t, J=6.5 Hz, 1H), 4.22 (d, J=3.0 Hz, 1H), 4.15(d, J=3.1 Hz, 1H), 4.11-3.48 (m, 38H), 3.27 (t, J=8.4 Hz, 1H), 3.19 (t,J=6.7 Hz, 2H), 2.78-2.71 (m, 3H), 2.51 (t, J=6.7 Hz, 2H), 2.00 (s, 3H),1.78 (t, J=12.1 Hz, 1H), 1.61 (q, J=7.1 Hz, 2H), 1.52 (q, J=7.1 Hz, 2H),1.39 (q, J=8.0 Hz, 2H).

C-3: Ac-Alkynyl_SSEA4-SH (RN=NHCOC₂H₄CCH, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.94 (d, J=3.9 Hz, 1H), 4.72 (d, J=8.4 Hz, 1H),4.58- 4.48 (m, 3H), 4.41 (t, J=6.5 Hz, 1H), 4.30-4.26 (m, 1H), 4.21 (d,J=3.1 Hz, 1H), 4.14 -3.54 (m, 37H), 3.32 (t, J=8.6 Hz, 1H), 3.24 (t,J=6.8 Hz, 2H), 2.83-2.74 (m, 3H), 2.59-2.49 (m, 5H), 2.43 (s, 1H), 2.06(s, 3H), 1.82 (t, J=12.1 Hz, 1H), 1.67 (p, J=6.9 Hz, 2H), 1.58 (p, J=6.9Hz, 2H), 1.48-1.38 (m, 2H).

C-4: Ac-Fluoride_SSEA4-SH (RN=NHCOCH₂F, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.90 (d, J=46.4 Hz, 2H), 4.95 (d, J=4.0 Hz, 1H),4.72 (d, J=8.5 Hz, 1H), 4.59-4.48 (m, 3H), 4.41 (t, J=6.6 Hz, 1H), 4.31-4.26 (m, 1H), 4.23 -4.18 (m, 1H), 4.14-3.54 (m, 36H), 3.36-3.29 (m, 1H),3.25 (t, J=6.8 Hz, 2H), 2.80 (m, 3H), 2.57 (t, J=6.7 Hz, 2H), 2.06 (s,3H), 1.84 (t, J=12.2 Hz, 1H), 1.67 (p, J=6.9 Hz, 2H), 1.58 (p, J=7.0 Hz,2H), 1.43 (q, J=8.3 Hz, 2H).

C-5: Ac-Phenyl_SSEA4-SH (RN=NHCOCH₂Ph, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.48-7.32 (m, 5H), 4.94 (d, J=3.6 Hz, 1H),4.73-4.68 (d, J=8.4 Hz, 1H), 4.52 (m, 3H), 4.41 (t, J=6.4 Hz, 1H),4.29-4.26 (m, 1H), 4.20 (d, J=3.0 Hz, 1H), 4.13-3.51 (m, 37H), 3.39 (dd,J=9.0, 1.8 Hz, 1H), 3.32 (t, J=8.6 Hz, 1H), 3.25 (t, J=6.7 Hz, 2H),2.83-2.74 (m, 3H), 2.56 (t, J=6.7 Hz, 2H), 2.04 (s, 3H), 1.80 (t, J=12.1Hz, 1H), 1.67 (q, J=7.2 Hz, 2H), 1.57 (q, J=7.1 Hz, 2H), 1.48-1.38 (m,2H).

C-6: Ac-Azido_SSEA4-SH (RN=NHCOCH₂N₃, R10=OH)

¹H NMR (400 MHz, D₂O) δ 4.88 (d, J=3.9 Hz, 1H), 4.66 (d, J=8.5 Hz, 1H),4.52- 4.43 (m, 3H), 4.36 (t, J=6.5 Hz, 1H), 4.22 (d, J=3.1 Hz, 1H), 4.14(d, J=3.1 Hz, 1H), 4.08-3.47 (m, 38H), 3.26 (t, J=8.4 Hz, 1H), 3.19 (t,J=6.8 Hz, 2H), 2.74 (m, 3H), 2.51 (t, J=6.7 Hz, 2H), 2.00 (s, 3H), 1.76(t, J=12.1 Hz, 1H), 1.61 (q, J=7.1 Hz, 2H), 1.53 (p, J=7.0 Hz, 2H), 1.38(q, J=8.3 Hz, 2H).

C-7: 5′-Azido_SSEA4-SH (RN=N₃, R10=OH)

¹H NMR (400 Hz, D₂O) δ 4.90 (d, J=4.0 Hz, 1H), 4.67 (d, J=8.4 Hz, 1H),4.51-4.46 (m, 3H), 4.37 (t, J=6.4 Hz, 1H), 4.24 (d, J=2.8 Hz, 1H), 4.15(d, J=2.8 Hz, 1H), 4.01-3.44 (m, 35H), 3.28 (t, J=8.4 Hz, 1H), 3.21 (t,J=6.8 Hz, 2H), 2.78-2.72 (m, 3H), 2.52 (t, J=7.2 Hz, 2H), 2.02 (s, 3H),1.77 (t, J=12.0 Hz, 1H), 1.67-1.60 (m, 2H), 1.58-1.50 (m, 2H), 1.43-1.37(m, 2H)

C-8: 9′-Azido_SSEA4-SH (RN=NHAc, R10=N3)

¹H NMR (400 MHz, D₂O) δ 4.90 (d, J=3.9 Hz, 1H), 4.68 (d, J=8.5 Hz, 1H),4.48 (dd, J=13.2, 7.9 Hz, 3H), 4.37 (t, J=6.5 Hz, 1H), 4.26-4.22 (m,1H), 4.16 (d, J=3.3 Hz, 1H), 4.09-3.44 (m, 36H), 3.31-3.24 (m, 1H), 3.20(t, J=6.8 Hz, 2H), 2.79-2.70 (m, 3H), 2.52 (t, J=6.7 Hz, 2H), 2.02 (d,J=2.0 Hz, 6H), 1.76 (t, J=12.1 Hz, 1H), 1.63 (p, J=6.9 Hz, 2H), 1.54 (p,J=6.9 Hz, 2H), 1.39 (q, J=8.3 Hz, 2H).

C-9: NHBz_SSEA4-SH (RN=NHBz, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.80-7.73 (m, 2H), 7.66-7.58 (m, 1H), 7.52 (dd,J=8.4, 7.0 Hz, 2H), 4.91 (d, J=3.9 Hz, 1H), 4.69 (d, J=8.5 Hz, 1H),4.57-4.44 (m, 3H), 4.38 (t, J=6.5 Hz, 1H), 4.27-4.22 (m, 1H), 4.18 (d,J=3.1 Hz, 1H), 4.16-3.52 (m, 36H), 3.29 (t, J=8.5 Hz, 1H), 3.20 (t,J=6.8 Hz, 2H), 2.84-2.72 (m, 3H), 2.52 (t, J=6.7 Hz, 2H), 2.03 (s, 3H),1.89 (t, J=12.2 Hz, 1H), 1.63 (p, J=6.8 Hz, 2H), 1.53 (q, J=7.1 Hz, 2H),1.40 (q, J=8.2 Hz, 2H).

D: Chemoenzymatic Synthesis of SSEA4 analog-allyl

SSEA4 analogs-allyl were synthesized via enzymatic regeneration strategyas described in Scheme5. In this system, ManNAc derivatives were reactedwith pyruvate and transformed into NeuSAc analogs by aldolase catalysis,followed by incorporation with Gb5-ally in the regeneration system (J.Am. Chem. Soc. 2013, 135, 14831-14839) to obtain the exemplary SSEA4analogs-allyl. (Scheme 5)

Detail of the reaction condition is described as follows: Gb5-allyl (18μmol), CTP (5 μmop, ManNAc derivative (27 μmol), sodium pyruvate (81μmol), PEP (55 μmol), and ATP (5 μmol), were dissolved in 50 mM Tris-HClbuffer (pH 8.0). Enzymesalpha-(2,3)-sialyltransferase (20 units), sialicacid aldolase(20 units) CMK (10 units), Pykf (10 units), PPA (10 units),and Pmcss (10 units) were added to the solution, and the reaction wasincubated at 37° C. for 8 hours and monitored by TLC plate. At the endof reaction, enzyme was denatured by heating at 100° C. for 5 minutes.The desired SSEA4-analog-allyl was purified by G25, DEAE, and SP column(80%).

¹H NMR of SSEA4 analogs-allyl

D-1. SSEA4-allyl (R1=OH, RN=NHAc, R10=OH)

¹H NMR (400 MHz, D₂O) δ 6.00 (m, 1H), 5.40-5.37 (d, J=17.3 Hz, 1H),5.30-5.28 (d, J=10.4 Hz, 1H), 4.92 (d, J=3.9 Hz, 1H), 4.70 (d, J=8.5 Hz,1H), 4.54-4.51 (m, 3H), 4.40-4.38 (m, 2H), 4.25-4.18 (m, 3H), 4.10-3.52(m, 34 H), 3.35-3.32 (t, J=8.6 Hz, 1H), 2.77 (dd, J=12.5, 4.6 Hz, 1H),2.03 (s, 6H), 1.80 (t, J=12.1 Hz, 1H)

D-2. Neu5Gc_SSEA4-allyl (R1=OH, RN=NHGc, R10=OH)

¹H NMR (400 MHz, D₂O) δ 5.99 (m, 1H), 5.38 (dd, J=1.2, 17.2 Hz, 1H),5.29 (dd, J=1.2, 10.0 Hz, 1H), 4.93 (d, J=4.0 Hz, 1H), 4.69 (d, J=8.4Hz, 1H), 4.58-4.51 (m, 3H), 4.43-4.37 (m, 2H), 4.28-4.17 (m, 3H),4.14-3.52 (m, 34 H), 3.33 (t, J=8.8 Hz, 1H), 2.77 (dd, J=4.8, 12.4 Hz,1H), 2.03 (s, 3H), 1.81 (t, J=12.0 Hz, 1H).

D-3. Ac-Fluoride_SSEA4-allyl (R1=OH, RN=NHCOCH₂F, R10=OH)

¹H NMR (400 MHz, D₂O) δ 5.96 (m, 1H), 5.36 (dd, J=1.6, 17.2 Hz, 1H),5.25 (dd, J=1.6, 10.4 Hz, 1H), 4.89 (d, J=46.4 Hz, 2H), 4.88 (d, J=3.6Hz, 1 H), 4.65 (d, J=8.4 Hz, 1H), 4.53-4.45 (m, 3H), 4.39-4.32 (m, 2H),4.22-3.51 (m, 37H), 3.30 (t, J=8.4 Hz, 1H), 2.73 (dd, J=4.4, 12.4 Hz,1H), 2.00 (s, 3H), 1.85 (t, J=12.4 Hz, 1H).

D-4. Ac-Phenyl_SSEA4-allyl (R1=OH, RN=NHCOCH₂Ph, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.45-7.34 (m, 5H), 6.02 (m, 1H), 5.42 (dd,J=1.2, 17.2 Hz, 1H), 5.32 (dd, J=1.2, 10.4 Hz, 1H), 4.94 (d, J=4.0 Hz,1H), 4.72 (d, J=8.4 Hz, 1H), 4.59-4.52 (m, 3H), 4.46-4.38 (m, 2H),4.30-3.50 (m, 38 H), 3.42-3.32 (m, 4H), 2.77 (dd, J=4.4, 12.8 Hz, 1H),2.05 (s, 3H), 1.90 (t, J=12.0 Hz, 1H).

D-5. Ac-Azido_SSEA4-allyl (R1=OH, RN=NHCOCH₂N₃, R10=OH)

¹H NMR (400 MHz, D₂O) δ 5.95 (m, 1H), 5.35 (dd, J=1.6, 17.2 Hz, 1H),5.25 (dd, J=1.2, 10.4 Hz, 1H), 4.88 (d, J=3.6 Hz, 1H), 4.65 (d, J=8.4Hz, 1H), 4.52-4.46 (m, 3H), 4.40-4.32 (m, 2H), 4.23-4.18 (m, 3H),4.12-3.50 (m, 36 H), 3.30 (t, J=5.6 Hz, 1H), 2.72 (dd, J=4.8, 12.8 Hz,1H), 2.00 (s, 3H), 1.84 (t, J=12.4 Hz, 1H).

D-6. 5′-Azido_SSEA4-allyl (R1=OH, RN=N₃, R10=OH)

¹H NMR (400 MHz, D₂O): 6 5.99 (m, 1H), 4.40 (dd, J=1.6, 17.2 Hz, 1H),5.29 (d, J=10.4 Hz, 1H), 4.92 (d, J=3.6 Hz, 1H), 4.70 (d, J=8.4 Hz, 1H),4.56-4.51 (m, 3H), 4.43-4.38 (m, 2H), 4.26 (d, J=3.6 Hz, 2H), 4.22 (d,J=6.4 Hz, 1H), 4.10-3.46 (m, 35H), 3.36-3.32 (m, 1H), 2.74 (dd, J=4.8,12.4 Hz, 1H), 2.04 (s, 3H), 1.79 (t, J=12.4 Hz); HRMS (ESI-TOF, M−H−)C₄₄H₇₁N₄O₃₃ calcd for 1183.4001, found 1183.4056.

D-7. 9′-Azido_SSEA4-allyl (R1=OH, RN=NHAc, R10=N₃)

¹H NMR (400 MHz, D₂O) δ 5.96 (m, 1H), 5.36 (dd, J=1.6, 17.3 Hz, 1H),5.26 (dd, J=1.6, 10.4 Hz, 1H), 4.90 (d, J=3.6 Hz, 1H), 4.68 (d, J=8.4Hz, 1H), 4.55-4.47 (m, 3H), 4.41-4.35 (m, 2H), 4.25-4.14 (m, 3H),4.10-3.41 (m, 34 H), 3.31 (t, J=6.8 Hz, 1H), 2.72 (dd, J=4.8, 12.8 Hz,1H), 2.02 (s, 3H), 1.79 (t, J=12.0 Hz, 1H).

D-8. NHBz_SSEA4-allyl (R1=OH, RN=NHBz, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.76-7.73 (m, 2H), 7.59 (m, 1H), 7.51-7.46 (m,2H), 5.90 (m, 1H), 5.30 (dd, J=1.6, 17.2 Hz, 1H), 5.25 (dd, J=1.6, 10.8Hz, 1H), 4.89 (d, J=3.6 Hz, 1H), 4.67 (d, J=8.8 Hz, 1H), 4.55-4.45 (m,3H), 4.39-4.38 (m, 2H), 4.24-3.50 (m, 34H), 3.30 (t, J=8.0 Hz, 1H), 2.77(dd, J=4.4, 12.4 Hz, 1H), 2.01 (s, 3H), 1.90 (t, J=12.4 Hz, 1H).

E: Oxidation reaction for SSEA4 analog-aldehyde

In certain exemplary embodiments, a stirred solution of the SSEA4analogs-allyl in methanol and H₂O was ozonolysis for 15 minutes under O₃gas atmosphere at −70° C. The reaction mixture was quenched by dimethylsulfide (Me₂S) and then the solution was evaporated in vacuo. Thedesired SSEA4 analogs-aldehyde was then purified by G15. (Scheme 6)

¹HNMR of SSEA4 analogs-aldehyde

E-1: SSEA4-aldehyde (RN=NHAc, R10=OH)

1H NMR (400 MHz, D₂O) δ 5.19 (t, J=4.9 Hz, 1H), 4.89 (d, J=3.9 Hz, 1H),4.66 (d, J=8.4 Hz, 1H), 4.54-4.45 (m, 3H), 4.36 (t, J=6.5 Hz, 1H),4.25-4.20 (m, 1H), 4.15 (d, J=3.1 Hz, 1H), 4.08-3.47 (m, 32H), 3.37-3.30(m, 1H), 2.73 (dd, J=12.4, 4.6 Hz, 1H), 2.00 (d, J=0.9 Hz, 6H), 1.76 (t,J=12.1 Hz, 1H).

E-2: Neu5Gc_SSEA4-aldehyde (RN=NHGc, R10=OH)

¹HNMR (400 MHz, D₂O) δ 5.20 (t, J=4.9 Hz, 1H), 4.91 (d, J=3.9 Hz, 1H),4.68 (d, J=8.5 Hz, 1H), 4.52 (dt, J=8.5, 4.5 Hz, 3H), 4.38 (t, J=6.5 Hz,1H), 4.27-4.22 (m, 1H), 4.17 (d, J=3.1 Hz, 1H), 4.13-3.51 (m, 34H),3.38-3.32 (m, 1H), 2.76 (dd, J=12.4, 4.6 Hz, 1H), 2.02 (s, 3H), 1.80 (t,J=12.1 Hz, 1H).

E-3: Ac-Fluoride_SSEA4-aldehyde (RN=NHCOCH₂F, R10=OH)

1H NMR (400 MHz, D₂O) δ 5.21 (t, J=4.9 Hz, 1H), 4.90 (d, J=46.4 Hz, 2H),4.69 (d, J=8.5 Hz, 1H), 4.52 (t, J=8.0 Hz, 3H), 4.38 (t, J=6.4 Hz, 1H),4.24 (d, J=3.1 Hz, 1H), 4.17 (d, J=3.2 Hz, 1H), 4.10-3.45 (m, 33H),3.40-3.32 (m, 1H), 2.78 (dd, J=12.4, 4.6 Hz, 1H), 2.03 (s, 3H), 1.81 (t,J=12.2 Hz, 1H).

E-4: Ac-Phenyl_SSEA4-aldehyde (RN=NHCOCH₂Ph, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.48-7.27 (m, 5H), 5.22 (t, J=4.9 Hz, 1H), 4.92(d, J=4.0 Hz, 1H), 4.69 (d, J=8.4 Hz, 1H), 4.56-4.49 (m, 3H), 4.39 (t,J=6.5 Hz, 1H), 4.26 (m, 1H), 4.18 (m, 1H), 4.10-3.45 (m, 34H), 3.43-3.34(m, 1H), 2.76 (dd, J=12.4, 4.6 Hz, 1H), 2.03 (s, 3H), 1.78 (t, J=12.3Hz, 1H).

E-5: Ac-Azido_SSEA4-aldehyde (RN=NHCOCH₂N₃, R10=OH)

¹H NMR (400 MHz, D₂O) δ 5.20 (t, J=4.9 Hz, 1H), 4.90 (d, J=3.9 Hz, 1H),4.68 (d, J=8.5 Hz, 1H), 4.54-4.48 (m, 3H), 4.38 (t, J=6.4 Hz, 1H), 4.24(d, J=3.1 Hz, 1H), 4.17 (d, J=3.1 Hz, 1H), 4.13-3.51 (m, 34H), 3.39-3.32(m, 1H), 2.75 (dd, J=12.4, 4.6 Hz, 1H), 2.02 (s, 3H), 1.79 (t, J=12.2Hz, 1H).

E-6: 9′-Azido_SSEA4-aldehyde (RN=NHAc, R10=N₃)

¹H NMR (400 MHz, D₂O) δ 5.20 (t, J=4.9 Hz, 1H), 4.91 (d, J=3.9 Hz, 1H),4.69 (d, J=8.5 Hz, 1H), 4.52 (t, J=8.0 Hz, 3H), 4.38 (t, J=6.4 Hz, 1H),4.24 (d, J=3.1 Hz, 1H), 4.17 (d, J=3.2 Hz, 1H), 4.10-3.45 (m, 32H),3.39-3.32 (m, 1H), 2.74 (dd, J=12.5, 4.6 Hz, 1H), 2.03 (d, J=2.1 Hz,6H), 1.77 (t, J=12.1 Hz, 1H).

E-7: NHBz_SSEA4-aldehyde (RN=NHBz, R10=OH)

¹H NMR (400 MHz, D₂O) δ 7.83-7.76 (m, 2H), 7.63 (t, J=7.3 Hz, 1H), 7.53(t, J=7.7 Hz, 2H), 5.21 (t, J=4.9 Hz, 1H), 4.92 (d, J=3.8 Hz, 1H), 4.70(d, J=8.5 Hz, 1H), 4.57-4.49 (m, 3H), 4.39 (t, J=6.5 Hz, 1H), 4.26 (d,J=3.1 Hz, 1H), 4.19 (d, J=3.3 Hz, 1H), 4.16-3.52 (m, 32H), 3.40-3.34 (m,1H), 2.82 (dd, J=12.4, 4.6 Hz, 1H), 2.04 (d, J=4.7 Hz, 3H), 1.87 (t,J=12.1 Hz, 1H).

Example 3 Synthesis of SSEA3/SSEA4 Analog CRM197-Conjugates ViaSulfo-EMCS Crosslink

General Methods:

Step A. To modify SSEA3 analog-NH2 or SSEA4 analog-NH2 into SSEA3analog- SH or SSEA4-analog-SH

To synthesize SSEA3/4 analog CRM197-conjugates, the amine-terminatedSSEA3/4 analogs were reacted with the DTSSP linker in PBS buffer (pH7.4) at room temperature. To monitor the pH value of solution by pHpaper, and add some NaOH solution to the solution when the solutionbecame neuter or acid. After the reaction was stirred at roomtemperature for 12 hours, DTT was added to the solution at roomtemperature. The solution was kept stirring at 40° C., and then thesolvent was removed under reduced pressure. The residue was purified byLH-20 column chromatography to give SSEA3/4 analog-SH.

Step B: To modify CRM197 into CRM197-maleimide.

After the salt of commercial CRM197 (1.0 mg) was removed via alternatedissolving in water and dialyzing (Amicon Ultra-0.5, 10 kDa,), theresidue was dissolved in PBS buffer (pH 6.5, 1.0 mL) and transferredinto a sample vial. Sulfo-EMCS (1.0 mg, 8.22×10⁻⁶ mol) was added to thesolution, and then the reaction was kept stirring at room temperaturefor 2 hours. The mixture was purified by Amicon Ultra-0.5 (10 kDa).After using MALDI-TOF to check the molecular weight and BCA assay tocalculate the amount of protein, the CRM197-maleimid was stored in PBSbuffer (pH 7.2, 1.0 mg/mL) for next step. According to the data ofMALDI-TOF, the amount of maleimid function groups could be calculated.For example, when the molecular weight of CRM197-maleimid was 61841, thenumbers of maleimide function groups on CRM197-maleimid were(61841-58326)/193=18.2.

Step C: The Synthesis of SSEA3/4 analog-CRM197 Conjugates

The CRM197-maleimids were dissolved in PBS buffer (pH 7.2, theconcentration was 1.0 mg/mL) and then different amount of SSEA3/4analog-SH (5.0 mg/mL in PBS buffer, pH 7.2) were added into thesolution. The mixtures were stirred at room temperature for 2 hours. TheSSEA3/4 analog-CRM197 conjugates were purified by using Amicon Ultra-0.5(10 kDa) to remove the nonreactive SSEA3/4 analog-SH and sodiumphosphate salt via dialysis. The obtained SSEA3/4 analog-CRM197conjugates could be characterized by MALDI-TOF analysis to determine thecarbohydrate incorporation rate. The nonreactive SSEA3/4 analog-SH couldbe recovered after reacting with DTT and purifying by LH-20 columnchromatography.

TABLE 1 Carbohydrate incorporation rate of SSEA4 analog with CRM-197 viaSulfo-EMCS Molecular weight (y) average Code Sugar after glycosylationincorporation rate M1 SSEA4 75465 8.84 M2 Neu5Gc_SSEA4 70750 5.83 M3Ac-Alkynyl_SSEA4 68965 5.94 M4 Ac-Fluoride_SSEA4 69190 4.59 M5Ac-Phenyl_SSEA4 75454 8.10 M6 Ac-Azido_SSEA4 70274 5.30 M79′-Azido_SSEA4 76596 9.87 M8 Glc-azido_SSEA4 73047 8.00

Example 4 Syntheses of SSEA4-Gc-CRM197 Conjugates Via Sulfo-EMCSCrosslink

Step A: To modify SSEA4-Gc-NH2 into SSEA4-Gc-SH

DTSSP (5.0 mg, 8.22×10⁻⁶ mol) was added to a flask of SSEA4-Gc-NH2 (5.0mg, 4.01×10⁻⁶ mol) in PBS buffer (pH 7.4, 1.0 mL) at room temperature.To monitor the pH value of solution by pH paper, NaOH (1 M/water) wasadded to the solution when the solution became neuter or acid. After thereaction was stirred at room temperature for 12 hours, DTT (5.0 mg,32.41×10⁻⁶ mol) was added to the solution at room temperature. Thesolution was kept stirring at 40° C. for 1 hour, and then the solventwas removed under reduced pressure. The residue was purified by LH-20column chromatography to give SSEA4-Gc-SH (5.0 mg, 93%)

Step B: To modify CRM197 into CRM197-maleimide.

After the salt of commercial CRM197 (1.0 mg) was removed via alternatedissolving in water and dialyzing (Amicon Ultra-0.5, 10 kDa,), theresidue was dissolved in PBS buffer (pH 6.5, 1.0 mL) and transferredinto a sample vial. Sulfo-EMCS (1.0 mg, 8.22×10⁻⁶ mol) was added to thesolution, and then the reaction was kept stirring at room temperaturefor 2 hours. The mixture was purified by Amicon Ultra-0.5 (10 kDa).After using MALDI-TOF to check the molecular weight and BCA assay tocalculate the amount of protein, the CRM197-maleimid was stored in PBSbuffer (pH 7.2, 1.0 mg/mL) for next step. According to the data ofMALDI-TOF, the amount of maleimid function groups could be calculated.For example, when the molecular weight of CRM197-maleimid was 61841, thenumbers of maleimide function groups on CRM197-maleimid were(61841-58326)/193=18.2.

The CRM197-maleimids were dissolved in PBS buffer (pH 7.2, theconcentration was 1.0 mg/mL) and then different amount of SSE4Gc-SH (5.0mg/mL in PBS buffer, pH 7.2) were added into the solution. The mixtureswere stirred at room temperature for 2 hours. The SSEA4-Gc-CRM197conjugates were purified by using Amicon Ultra-0.5 (10 kDa) to removethe nonreactive SSEA4-Gc-SH and sodium phosphate salt via dialysis. Theobtained SSEA4-Gc-CRM197 conjugates could be characterized by MALDI-TOFanalysis to determine the carbohydrate incorporation rate as showing inTable 2. The nonreactive SSEA4-Gc-SH could be recovered after reactingwith DTT and purifying by LH-20 column chromatography.

Step C: To trap the nonreactive maleimides of CRM197-maleimide

The SSEA4-Gc-CRM197 conjugates were dissolved in PBS buffer (pH 7.2, theconcentration was 1.0 mg/mL) and 10.0 equivalent of 2-mercaptoethanol (5mg/mL, PBS buffer, pH 7.2) were added to the solution. The mixtures werestirred at room temperature for 2 hours. The SSEA4-Gc-CRM197 conjugateswere purified by using Amicon Ultra-0.5 (10 kDa) to remove thenonreactive 2-mercaptoethanol and sodium phosphate salt via dialysis andthen lyphophilized to a white powder.

TABLE 2 Conjugation of CRM197 with SSEA4-Gc Number Amount of NumberHSC₂H₄OH CRM197 of Linkers PBS Buffer SSEA4-Gc Reaction of (5 mg/mL)CRM197 (μg) Linkers (mol) (pH 7.4, μL) (5 mg/mL) Time Sugars 10.0 eq.(μg) 1 1388 21.9 5.21 × 10⁻⁷ 1000 28.1 μL 2 hr 2.0 81.4 μL 1550.4 (0.2eq.) 2 694 21.9 2.61 × 10⁻⁷ 500 28.2 μL 2 hr 4.2 40.8 μL 657.4 (0.4 eq.)3 694 21.9 2.61 × 10⁻⁷ 500 56.4 μL 2 hr 6.5 40.8 μL 665.0 (0.8 eq.) 4694 21.9 2.61 × 10⁻⁷ 500 84.5 μL 2 hr 6.9 40.8 μL 627.0 (1.2 eq.) 5 69421.9 2.61 × 10⁻⁷ 500 140.9 μL 2 hr 7.1 40.8 μL 615.6 (2.0 eq.) 6 69421.9 2.61 × 10⁻⁷ 500 281.8 μL 2 hr 7.0 40.8 μL 665.0 (4.0 eq.) 7 69421.9 2.61 × 10⁻⁷ 500 704.4 μL 2 hr 6.8 40.8 μL 695.4 (10.0 eq.) a) M.W.of CRM197 = 58326 → 1000 μg = 0.1715 × 10⁻⁷ mol b) M.W. of SSEA4-Gc-SH =1349.479 → 5 mg/mL = 37.051 × 10⁻⁷ mol/mL c) M.W. of 2-Mercaptoethanol =78.13 → 5 mg/mL = 639.91 × 10⁻⁷ mol/mL

Example 5 SSEA4 Analog-CRM197 Conjugate Via SBAP Crosslink

CRM-197 was dissolved in 0.1 M phosphate buffer pH 7.4 (˜1 mg/ml), andSBAP (1.0 mg) was added to the solution. The solution was stirred gentlyfor 2 hrs at room temperature. The mixture was then diluted with PBSbuffer and centrifuge against 5 changes of 0.1 M phosphate buffer pH 7.4by Amicon Ultra-0.5 (10 kDa, 2×). The obtained modified CRM-197 can becharacterized by MALDI-TOF (positive mode, matrix was sinapinic acid,H₂O) analysis to determine the SBAP incorporation rate.

Modified CRM-197 was dissolved in 0.1 M phosphate buffer pH 8.0 (˜1mg/ml), and SSEA4-SH analog was added to the solution. The mixture wasstirred for 1 day at room temperature. The mixture was then diluted withPBS buffer and centrifuge against 5 changes of 0.1 M PBS buffer pH 7.4by Amicon Ultra-0.5 (10 kDa, 2×). The obtained sugar-protein conjugatecould be characterized by MALDI-TOF (positive mode, matrix was sinapinicacid, H₂O) analysis to determine the carbohydrate incorporation rate.(Scheme 9)

TABLE 3 Carbohydrate incorporation rate of SSEA4 analog with CRM-197 viaSBAP Molecular weight (x) average Code Sugar after glycosylationincorporation rate S1 SSEA4 68212 4.79 S2 Neu5Gc_SSEA4 67651 4.84 S3Ac-Alkynyl_SSEA4 70308 5.70 S4 Ac-Fluoride_SSEA4 69309 5.01 S5Ac-Phenyl_SSEA4 68891 5.05 S6 Ac-Azido_SSEA4 68359 4.50 S75′-Azido_SSEA4 71638 7.06 S8 9′-Azido_SSEA4 72545 7.90 S9Glc-azido_SSEA4 67131 3.9 S10 NHBz_SSEA4 69636 5.50

Example 6 SSEA4 Analog-CRM197 Conjugate Via Reductive AminationCrosslink

In certain embodiments, CRM197 was dissolved in 0.1 M phosphate buffer(pH 6-9) (˜1 mg/ml), and enough quantity SSEA4-aldehyde analogs andNaCNBH3 were added to the solution. The solution was stirred gently for3 days at room temperature. The mixture was then diluted with deionizedwater and centrifuge against 5 changes of 0.1 M phosphate buffer pH 7.4by Amicon Ultra-0.5 (10 kDa, 2×). The obtained sugar-protein conjugatewas characterized by MALDI-TOF (positive mode, matrix was sinapinicacid, H₂O) analysis to determine the carbohydrate incorporation rate.(Scheme 10)

TABLE 4 Carbohydrate incorporation rate of SSEA4 analog with CRM-197 viareductive amination Molecular weight (m) average Code Sugar afterglycosylation incorporation rate R1 SSEA4 69025 8.89 R2 Neu5Gc_SSEA465154 5.6 R3 Ac-Fluoride_SSEA4 69315 9 R4 Ac-Phenyl_SSEA4 71329 10.1 R5Ac-Azido_SSEA4 67765 7.6 R6 9′-Azido_SSEA4 67635 7.58 R7 NHBz_SSEA467124 6.95

Example 7 Immunization Determination of the SSEA4 Analog-CRM197Conjugates

Exemplary Method

To demonstrate the efficacy/immunogenicity of the SSEA4 analog CRM197conjugates (S1˜S10), female C57BL/6 mice (n=5 for each group) werevaccinated intramuscularly with 0.5 μg of SSEA4 analog CRM197-conjugatescombining the use of 2.0 μg of glycolipid adjuvant. Control mice weregiven only phosphate buffer saline with 2.0 μg of glycolipid adjuvant.The vaccination was conducted at biweekly intervals for 2 months, andthe antisera from the immunized mice were collected one week after eachvaccination. The antibody titers against SSEA4 were examined by ELISAusing SSEA4 immobilized 96-well titer plates. ELISA was conducted usingSSEA4 immobilized 96-well titer plate. Briefly, the diluted antiserawere incubated with the immobilized SSEA4 at room temperature for 2 hr.After the washing cycle, the captured anti-SSEA4 antibodies were thendetected using HPR-conjugated anti-IgG or IgM specific antibody.

To determine if the glycan-protein conjugation method would interferethe immune response, native SSEA4 was conjugated with CRM197 throughEMCS linker (M1), SBAP linker (S1) or reductive amination (R1) and usedfor immunogenicity study as described above.

Representative Result

After four times of immunization, native SSEA4, as well as all eightSSEA4 analogs, could positively elicit both IgG (FIG. 3A) and IgM (FIG.3B) antibodies against SSEA4 when combining the use of Gal-C34 adjuvant.There is no significant difference in the titers of anti-SSEA4 IgG andIgM antibodies among different analog groups. In addition, Glc-C34 canalso be used as vaccine adjuvant for inducing both IgG (FIG. 4A) and IgM(FIG. 4B) antibodies against SSEA4 when co-administering with nativeSSEA4 and the other analogs.

Furthermore, the results shown in FIG. 5 indicated that theglycan-protein conjugation method can affect the immune response.Combining the use of Gal-C34, the SSEA4-EMCS-CRM197 (M1) elicited ahigher anti-SSEA4 IgG antibody titer when comparing to SSEA4-SBAP-CRM197(S1) and SSEA4-CRM197 (conjugated through reductive amination, R1).

Example 8 Immunogenicity Study of the SSEA4 Analogs CRM197-Conjugates

To demonstrate the immunogenicity of the SSEA4 analog CRM197-conjugates,five female BALB/c mice were immunized intramuscularly with 2 μg ofSSEA4 analog CRM197-conjugates and 2 μg of the glycolipid adjuvant C34three times at biweekly intervals. In the previous study, the anti-GHantibodies titer was low with SSEA4 analog-protein conjugates alonewithout any adjuvants. The antisera from each immunogen were obtainedten days after the third immunization and were tested on the glycanmicroarray containing 94 chemically synthesized glycans, including globoseries glycans and other tumor-associated carbohydrate antigens. Becausesome chemical modifications were carried out on the glycan, somefunctional linkers were also included in the glycan array to check thecross reactivity.

Antibodies induced by the SSEA4-Gc CRM197-conjugates were specificallyrecognized by SSEA4-Gc, native SSEA4 or SSEA4 tetrasaccharide fragmentsbut not by other TACAs and functional linkers. The sera obtained fromthe glycoconjugates induced high IgG antibody titers, indicating aT-cell-dependent immune response. Interestingly, no significant IgMproduction was observed for SSEA4-Gc or native SSEA4. Regarding the IgGlevel against GloboH, the titers of antibodies induced by SSEA4-GcCRM197 was much higher than the nature form native SSEA4-CRM197conjugate. Among them the 6.9 molecule of SSEA4-Gc conjugated with onemolecule of CRM197 can induce the highest antibody titers.

Mice Dosage and Immunization Schedule

For comparing the immunogenicity of SSEA4 analog CRM197, ten groups offive mice (8-week-old female Balb/c mice, BioLASCO, Taiwan) wereimmunized intramuscularly with glycolipid C34. Three immunizations weregiven at 2-week intervals. Each vaccination contained 2 μg SSEA4 analogand 2 μg C34. Control mice were injected with phosphate buffer saline(PBS). Mice were bled before the first immunization (preimmune) and 10days after the third immunization. All of the sera were obtained bycentrifugation at 4,000×g for 10 min. The serologic responses wereanalyzed by glycan microarray.

Serologic Assay with Glycan Array

Mouse sera were diluted with 1% BSA/PBST buffer (PBST buffer: PBS and0.05% Tween-20, pH 7.4). The glycan microarray was blocked withSuperblock blocking buffer (Pierce) for 1 h at 4° C. and washed threetimes with PBST buffer before use. The serum dilutions were thenintroduced to the glycan microarray and incubated at 4° C. for 1 h.Excess serum antibodies were washed out and the microarrays wereincubated individually with Alexa Fluor 647-conjugated goat anti-mouseIgG antibody or DyLight 649-conjugated goat anti-mouse IgM antibody asthe 2nd antibody at 4° C. in dark for 1 h. The slides were then washedthree times with PBST and scanned at 635 nm wavelength with a microarrayfluorescence chip reader (GenePix 4300A; Molecular Devices Corporation)and scanned images were analyzed with GenePix Pro-6.0 analysis software(Axon Instruments, Union City, Calif., USA).

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features. From the above description, one skilled in the art caneasily ascertain the essential characteristics of the describedembodiments, and without departing from the spirit and scope thereof,can make various changes and modifications of the embodiments to adaptit to various usages and conditions. Thus, other embodiments are alsowithin the claims.

1. An immunogenic composition comprising: (a) a glycan conjugateincluding a carrier and one or more glycans, and optionally (b) anadjuvant; wherein each of the one or more glycans is conjugated with thecarrier through a linker having the formula (III):

wherein: X₁ is —OR or —SR, wherein R is an oxygen or sulfur protectinggroup, optionally substituted C₁₋₁₀ alkyl, optionally substituted aryl,optionally substituted acyl, or optionally substituted imidoyl; eachinstance of R¹, R², R³, R⁴, R⁵, R⁶ and L is independently selected fromhydrogen, halogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedheterocyclyl, optionally substituted aryl, —N₃, —NO₂, —N(R^(B))₂,—N(R^(A))C(O)R^(A), —OR^(A), —OC(O)R^(A), —CR^(A), —C(O)N(R^(B))₂, —CN,—C(O)R^(A), —C(O)OR^(A), —S(O)R^(A), —SO₂R^(A), —SO₂N(R^(B))₂, and—NHSO₂R^(B); each instance of R^(A) is independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted heterocyclyl, andoptionally substituted aryl; each instance of R^(B) is independentlyselected from hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, and optionally substituted aryl; and providedthe glycan conjugate is not of the formula (III-a) and (III-b):


2. The immunogenic composition of claim 1, wherein L is —OH.
 3. Theimmunogenic composition of claim 2, wherein at least one instance of R¹,R², R³, R⁴, R⁵ and R⁶ is —N₃.
 4. The immunogenic composition of claim 2,wherein at least one instance of R¹, R², R³, R⁴, R⁵ and R⁶ is —F.
 5. Theimmunogenic composition of claim 1, wherein L is of the formula:

wherein: each instance of R⁸, R⁹, R¹⁰ and R¹¹ is independently selectedfrom hydrogen, halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted aryl, —N₃, —NO₂,—N(R^(B))₂, —N(R^(A))C(O)R^(A), —OR^(A), —OC(O)R^(A), —CR^(A),—C(O)N(R^(B))₂, —CN, —C(O)R^(A), —C(O)OR^(A), —S(O)R^(A), —SO₂R^(A),—SO₂N(R^(B))₂, and —NHSO₂R^(B); wherein R¹² is H, OH, or halogen; R_(N)is selected from —N₃, —NO₂, —N(R^(B))₂, —N(R^(A))C(O)R^(A), —OR^(A),—OC(O)R^(A), —SR^(A), —C(O)N(R^(B))₂, —CN, —C(O)R^(A), —C(O)OR^(A),—S(O)R^(A), —SO₂R^(A), —SO₂N(R^(B))₂, and —NHSO₂R^(B); each instance ofR^(A) is independently selected from hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocyclyl, and optionally substituted aryl;each instance of R^(B) is independently selected from hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted heterocyclyl, and optionallysubstituted aryl.
 6. The immunogenic composition of claim 5, wherein theglycan conjugate is of formula (IV):


7. The immunogenic composition of claim 6, wherein at least one instanceof R¹, R², R³, R⁸, R⁹, R¹⁰, and R¹¹ is —N₃.
 8. The immunogeniccomposition of claim 6, wherein at least one instance of R², R³, R⁸, R⁹,R¹⁰, and R¹¹ is —F.
 9. The immunogenic composition of claim 1, whereinthe carrier is a protein, a lipid, a lipolized protein, a virus, apeptide, or a dendrimer of glycopeptides.
 10. The immunogeniccomposition of claim 9, wherein the carrier is a protein selected fromthe group consisting of tetanus toxoid (TT), diphtheria toxoid (DT),diphtheria toxin cross-reacting material ₁₉₇ (CRM₁₉₇), fragment C of TT,Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), protein D,outer-membrane protein (OMP) and pneumolysin.
 11. The immunogeniccomposition of claim 10, wherein the carrier protein is selected fromthe group consisting of TT, DT and CRM_(197.)
 12. The immunogeniccomposition of claim 11, wherein the glycan conjugate is of the formula(IV-a) or (IV-b):

wherein m is an integer of 1 to 40, inclusive.
 13. The immunogeniccomposition of claim 1, wherein the linker is a hetero- orhomo-bifunctional linker.
 14. The immunogenic composition of claim 1,wherein the adjuvant is a glycolipid capable of binding a CD1d moleculeon a dendritic cell.
 15. The immunogenic composition of claim 1, whereinthe adjuvant is C34, 7DW8-5, C17, C23, Gluco-C34, Aluminum salt,Squalene, MF59, or QS-21.
 16. The immunogenic composition of claim 1,wherein the immunogenic composition is capable of eliciting an immuneresponse against a cancer cell.
 17. The immunogenic composition of claim16, wherein the cancer cell is selected from the group consisting of abrain cancer cell, a lung cancer cell, a breast cancer cell, an oralcancer cell, an esophagus cancer cell, a stomach cancer cell, a livercancer cell, a bile duct cancer cell, a pancreatic cancer cell, a coloncancer cell, a kidney cancer cell, a bone cancer cell, a skin cancercell, a cervical cancer cell, an ovarian cancer cell, and a prostatecancer cell.
 18. The immunogenic composition of claim 16, wherein theimmune response includes generation of antibodies that specifically bindto one or more of the antigens selected from the group consisting ofSSEA3 and SSEA4.
 19. The immunogenic composition of claim 18, whereinthe antibodies neutralize one or more of SSEA3 and SSEA4 antigenexpressed on the surface of cancer cells or cancer stem cells.
 20. Theimmunogenic composition of claim 18, wherein the antibodiespredominantly includes IgG antibodies.
 21. A cancer vaccine, comprisinga therapeutically effective amount of the immunogenic composition ofclaim 1 and a pharmaceutically acceptable excipient.
 22. The cancervaccine of claim 21, wherein the cancer vaccine is able to induce ananti-cancer immune response in a subject.
 23. A method of treatingcancer in a subject in need thereof wherein the method comprisingadministering a therapeutically effective amount of the immunogeniccomposition of claim
 1. 24. The method of claim 23 wherein the vaccineis co-administered in combination with another therapeutic agent. 25.The method of claim 23, wherein the cancer is selected from the groupconsisting of brain cancer, lung cancer, breast cancer, oral cancer,esophageal cancer, stomach cancer, liver cancer, bile duct cancer,pancreatic cancer, colon cancer, kidney cancer, bone cancer, skincancer, cervical cancer, ovarian cancer, and prostate cancer.
 26. Themethod of claim 25, wherein the cancer cell expresses SSEA3 and/or SSEA4antigen on the surface of the cell.
 27. The method of claim 26, whereinthe subject is a human.
 28. The method of claim 23, wherein theimmunogenic composition or the cancer vaccine is administeredsubcutaneously.
 29. An isolated monoclonal antibody and/or a bindingfragment thereof raised against the immunogenic composition of claim 1.30. A composition comprising an effective amount of the antibody orantigen-binding fragment of claim 29 and a pharmaceutically acceptablecarrier.
 31. A method for making the immunogenic composition of claim 1.32. The method of claim 31 wherein the method comprising: providing acarrier; conjugating one or more glycan to the carrier by conjugationreaction; wherein each of the one or more glycan is SSEA3 or SSEA4. 33.An immunogenic composition comprising multivalent construct targetingone or more of SSEA4 and SSEA3 and their analogs thereof wherein theglycans are linked to a template and a carrier,

wherein n can be an integer from 1 to 10; wherein glycan can be selectedfrom the group consisting of Formulas I, II, III, and IV; wherein if nis 2 or more, each glycan can be the same as another glycan on theaspartyl peptide or a difference glycan on the aspartyl peptide.
 34. Thecomposition of claim 33 wherein the glycan is selected from the groupconsisting of SSEA3 and SSEA4.
 35. The composition of claim 33 whereinthe multivalent construct has the structure:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and L on each glycan moiety can be thesame or different.
 36. A compound having the formula (I):

or a salt thereof, wherein: X₁ is —OR or —SR, wherein R is hydrogen, aoxygen or sulfur protecting group, optionally substituted C₁₋₁₀ alkyl,optionally substituted aryl, optionally substituted acyl, or optionallysubstituted imidoyl; each instance of R¹, R², R³, R⁴, R⁵, R⁶ and L isindependently selected from hydrogen, halogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocyclyl, optionally substituted aryl, —N₃,—NO₂, —N(R^(B))₂, —N(R^(A))C(O)R^(A), —OR^(A), —OC(O)R^(A), —SR^(A),—C(O)N(R^(B))₂, —CN, —C(O)R^(A), —C(O)OR^(A), —S(O)R^(A), —SO₂R^(A),—SO₂N(R^(B))₂, and —NHSO₂R^(B); each instance of R^(A) is independentlyselected from hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, and optionally substituted aryl; each instanceof R^(B) is independently selected from hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocyclyl, and optionally substituted aryl;and provided the compound is not of the formula (I-a) or (I-b):


37. The compound of claim 35, wherein L is —OH.
 38. The compound ofclaim 35, wherein L is of the formula:

wherein: each instance of R⁸, R⁹, R¹⁰ and R¹¹ is independently selectedfrom hydrogen, halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted aryl, —N₃, —NO₂,—N(R^(B))₂, —N(R^(A))C(O)R^(A), —OR^(A), —OC(O)R^(A), —SR^(A),—C(O)N(R^(B))₂, —CN, —C(O)R^(A), —C(O)OR^(A), —S(O)R^(A), —SO₂R^(A),—SO₂N(R^(B))₂, and —NHSO₂R^(B); wherein R¹² is H, OH, or halogen; R_(N)is selected from —N₃, —NO₂, —N(R^(B))₂, —N(R^(A))C(O)R^(A), —OR^(A),—OC(O)R^(A), —SR^(A), —C(O)N(R^(B))₂, —CN, —C(O)R^(A), —C(O)OR^(A),—S(O)R^(A), —SO₂R^(A), —SO₂N(R^(B))₂, and —NHSO₂R^(B); each instance ofR^(A) is independently selected from hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocyclyl, and optionally substituted aryl;and each instance of R^(B) is independently selected from hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted heterocyclyl, and optionallysubstituted aryl.
 39. The compound of claim 35, wherein the compound isof Formula (II):


40. The compound of claim 39, wherein at least one instance of R¹, R²,R³, R⁸, R⁹, R¹⁰ and R¹¹ is —F.
 41. The compound of claim 39, wherein atleast one instance of R¹, R², R³, R⁸, R⁹, R¹⁰ and R¹¹ is —N₃.
 42. Themethod of treating hyperproliferative disease or condition comprisingthe administering to a subject in need thereof a therapeuticallyeffective amount of the compound of claim 36.